In dieser Präsentation erfahren Sie Alles über die Anwendungsmöglichkeiten der Geldokumentations-Systeme der Firma Syngene: Lighting and Filter Combinations, Examples, Chemiluminescence versus Fluorescence Western blot detection, Film versus digital imaging Chemiluminescence Western blot, Dot Blot Protocol, Fluorescent Western blot general protocol, General Tips and advice for Fluorescence Western blots, General Tips and advice for IR Western blots. Sprechen Sie uns an unter info@labortechnik.com oder besuchen Sie uns auf unserer Website https://www.labortechnik.com/de .
This document outlines the development of an aptamer-based MRI contrast agent for thrombin detection. The goals of the project are to synthesize and purify an aptamer-chelate conjugate and screen contrast agents to find the best system for measuring thrombin. Initial results showed successful aptamer synthesis but mass spectrometry revealed the conjugation reaction was not efficient. Future work will involve reacting the aptamer with DTPA or DOTA chelates and testing the conjugates in MRI.
The resolution and performance of an optical microscope can be characterized by a quantity known as the modulation transfer function (MTF), which is a measurement of the microscope's ability to transfer contrast from the specimen to the intermediate image plane at a specific resolution.
A Beginner’s Guide to the Principles and Applications of FRETExpedeon
FRET, or Fluorescence Resonance Energy Transfer, was first described more than 50 years ago. The availability of new dyes and detection technology has resulted in a much wider use of the application in recent years, especially in biomedical R&D.
Handbook of Optical Filters for Fluorescence Microscopychromatechnology
This guide is a compilation of the principles and know-how that the engineers at Chroma Technology Corp use to design filters for a variety of fluorescence microscopes and applications, including wide-field microscopes, confocal microscopes, and applications involving simultaneous detection of multiple fluorescent probes. Also included is information on the terms used to describe and specify optical filters and practical information on how filters can affect the optical alignment of a microscope. Finally, the handbook ends with a glossary of terms that are italicized or in boldface in the text.
The document discusses inkjet printing of functional materials on plasma treated surfaces. It explores using a μPlasma printhead to selectively treat substrate surfaces, improving their wetting behavior and allowing for more precise inkjet printing. Experiments showed that plasma treatment increased surface energy and wettability over time. Small plasma spots around 1mm in diameter were achieved and enabled localized inkjet printing within the treated areas at high resolution. A design of experiments approach was used to model how plasma spot size could be influenced by varying print height and number of treatments.
The document discusses various factors that affect image quality in nuclear medicine imaging, including spatial resolution, contrast, and noise. It describes methods for evaluating spatial resolution such as using bar phantoms or line spread functions. Modulation transfer functions can also be used to characterize spatial resolution and compare different imaging systems. Image contrast and noise are affected by factors like radiopharmaceutical uptake, scatter radiation, and count rates. Quality assurance tests are important for ensuring optimal system performance and image quality.
This document discusses optical coherence tomography (OCT), including its working principles, light sources used, and comparisons to other imaging modalities. OCT uses low coherence interferometry to perform high resolution, cross-sectional imaging of biological tissues. The document describes time domain OCT which scans the reference arm, and spectral domain OCT which uses a spectrometer instead. OCT has advantages over other modalities like ultrasound and MRI in providing micrometer-scale resolution and millimeter-scale imaging depths without the use of ionizing radiation.
Computed radiography and direct/digital radiography are two digital imaging techniques. Computed radiography uses an imaging plate that captures x-ray data, which is then converted to a digital image. Direct digital radiography uses detectors like TFT or flat panel detectors to directly capture x-ray data digitally. Both techniques offer benefits over traditional film like faster imaging and easier sharing of images.
This document outlines the development of an aptamer-based MRI contrast agent for thrombin detection. The goals of the project are to synthesize and purify an aptamer-chelate conjugate and screen contrast agents to find the best system for measuring thrombin. Initial results showed successful aptamer synthesis but mass spectrometry revealed the conjugation reaction was not efficient. Future work will involve reacting the aptamer with DTPA or DOTA chelates and testing the conjugates in MRI.
The resolution and performance of an optical microscope can be characterized by a quantity known as the modulation transfer function (MTF), which is a measurement of the microscope's ability to transfer contrast from the specimen to the intermediate image plane at a specific resolution.
A Beginner’s Guide to the Principles and Applications of FRETExpedeon
FRET, or Fluorescence Resonance Energy Transfer, was first described more than 50 years ago. The availability of new dyes and detection technology has resulted in a much wider use of the application in recent years, especially in biomedical R&D.
Handbook of Optical Filters for Fluorescence Microscopychromatechnology
This guide is a compilation of the principles and know-how that the engineers at Chroma Technology Corp use to design filters for a variety of fluorescence microscopes and applications, including wide-field microscopes, confocal microscopes, and applications involving simultaneous detection of multiple fluorescent probes. Also included is information on the terms used to describe and specify optical filters and practical information on how filters can affect the optical alignment of a microscope. Finally, the handbook ends with a glossary of terms that are italicized or in boldface in the text.
The document discusses inkjet printing of functional materials on plasma treated surfaces. It explores using a μPlasma printhead to selectively treat substrate surfaces, improving their wetting behavior and allowing for more precise inkjet printing. Experiments showed that plasma treatment increased surface energy and wettability over time. Small plasma spots around 1mm in diameter were achieved and enabled localized inkjet printing within the treated areas at high resolution. A design of experiments approach was used to model how plasma spot size could be influenced by varying print height and number of treatments.
The document discusses various factors that affect image quality in nuclear medicine imaging, including spatial resolution, contrast, and noise. It describes methods for evaluating spatial resolution such as using bar phantoms or line spread functions. Modulation transfer functions can also be used to characterize spatial resolution and compare different imaging systems. Image contrast and noise are affected by factors like radiopharmaceutical uptake, scatter radiation, and count rates. Quality assurance tests are important for ensuring optimal system performance and image quality.
This document discusses optical coherence tomography (OCT), including its working principles, light sources used, and comparisons to other imaging modalities. OCT uses low coherence interferometry to perform high resolution, cross-sectional imaging of biological tissues. The document describes time domain OCT which scans the reference arm, and spectral domain OCT which uses a spectrometer instead. OCT has advantages over other modalities like ultrasound and MRI in providing micrometer-scale resolution and millimeter-scale imaging depths without the use of ionizing radiation.
Computed radiography and direct/digital radiography are two digital imaging techniques. Computed radiography uses an imaging plate that captures x-ray data, which is then converted to a digital image. Direct digital radiography uses detectors like TFT or flat panel detectors to directly capture x-ray data digitally. Both techniques offer benefits over traditional film like faster imaging and easier sharing of images.
Flat Panel Digital Fluoroscopy by Akash Das AkashDas169
This document discusses flat panel digital fluoroscopy. It begins by explaining that fluoroscopy is a technique that uses a fluoroscope to obtain real-time moving images of a patient's internal structures. It then classifies fluoroscopy into C-arm and under/over table units, as well as conventional and digital units. The document goes on to explain that flat panel detectors have replaced image intensifier tubes and CCD cameras in digital fluoroscopy. Flat panel detectors consist of an array of individual detector elements and use a CsI phosphor, amorphous silicon, and thin film transistors to convert X-rays into light and then electronic signals. In closing, the mechanisms of flat panel detectors and traditional image intensifiers are compared through diagrams
Gel documentation systems are used to image and document nucleic acid and protein gels stained with ethidium bromide. They typically consist of a UV light source, hood, and camera. Ethidium bromide fluorescence allows visualization of DNA/RNA bands under UV light. Larger amounts of nucleic acid bind more ethidium bromide and appear brighter. Gel doc systems are used to capture and analyze gel images for applications like electrophoresis, blotting, and colony counting. Components include the camera, lenses, illuminator, stage, and software for image processing and analysis.
Chromatography is a technique used to separate mixtures by differential distribution between a stationary and mobile phase. It has various applications in science and medicine. The document discusses the history of chromatography, types including thin layer chromatography, gas chromatography, high performance liquid chromatography, and modes such as adsorption, partition, ion exchange, and size exclusion chromatography. Chromatography is commonly used for analytical and preparative purposes in various fields.
The document summarizes the key components and parameters of fluoroscopy systems. It discusses the image intensifier, which converts x-ray photons into light photons and uses electrodes to focus electrons onto an output screen. Parameters like conversion coefficient, brightness uniformity, and spatial resolution are described. It also covers the image intensifier's connection to a TV system using cameras like vidicons or CCDs, and how this produces a video signal to display fluoroscopy images on a monitor in real-time.
This document discusses different types of radiography detectors, focusing on radiographic film. It describes the components and construction of radiographic film, including the base, emulsion, and silver halide crystals. It explains how x-rays or light from intensifying screens interact with the emulsion to create a latent image. Key factors for selecting radiographic film types are also summarized, such as contrast, speed, resolution, crossover effects, spectral matching of screens and film, and safelighting considerations.
Computed radiography (CR) uses reusable imaging plates and associated hardware and software to acquire and display digital x-ray images as an alternative to traditional film-based radiography. The document provides an overview of the key components of a CR system, including the imaging plate, reader/digitizer, and workstation. It describes how a latent image is captured and stored in the phosphor plate from x-ray exposure, then stimulated and converted to a digital image by the reader using a laser. The advantages of CR over conventional radiography are also summarized, such as reusability of plates and improved image manipulation, storage and sharing capabilities.
Pharmaceutical imaging techniques provide visual representations of objects like body parts or pharmaceutical products for quality control, data collection, or disease diagnosis using computerized methods like ultrasound or spectroscopy. Imaging technologies are gaining attention in the pharmaceutical industry due to their potential to accelerate drug discovery and development. Common techniques discussed in the document include chemical imaging, elemental imaging, digital imaging, fluorescence correlation spectroscopy, and micro-X-ray fluorescence, which are used for applications like content uniformity testing, particle characterization, counterfeit detection, and detecting drug distribution.
This document discusses various concepts related to radiographic image quality and measurements. It defines terms like radiographic contrast, spatial resolution, contrast resolution, noise, and artifacts. It describes how factors like the film, geometry, and subject can impact radiographic quality. It also discusses optical density, sensitometry, and how the characteristic curve relates exposure to density. The modulation transfer function and how it relates to spatial frequencies is explained. Overall, the document provides an overview of key technical factors and measurements that influence the quality of radiographic images.
In vivo confocal microscopy allows for high-resolution imaging of the living cornea. It works by scanning a small region of the cornea with light to build up an image point-by-point, achieving high magnification without disrupting corneal tissue. There are three main types: tandem scanning uses a Nipkow disk, scanning slit uses a slit aperture, and laser scanning confocal microscopy is commonly used to serially image thin corneal layers. Confocal microscopy can be used to diagnose and monitor conditions like infections, dystrophies, and thinning disorders by visualizing corneal cells, structures, deposits and infiltrates. It provides rapid diagnosis and guidance for treatment.
Dharmasekara is an energetic and knowledgeable professional with a Bachelor's degree in Finance. He has over 5 years of experience working as an Audit Supervisor at KPMG Sri Lanka, where he has provided assurance, audit, and consulting services to clients in various industries. He is proficient in English, Sinhala, accounting standards, and computer programs. Dharmasekara seeks new opportunities to utilize his skills in finance, auditing, and client services.
This document defines color and describes three key properties: hue, intensity, and value. It introduces the color wheel and primary, secondary, and tertiary colors. Different color schemes are explained including complementary, analogous, triadic, split-complementary, rectangle, and square. Color temperature is defined as describing the overall warmth or coolness of colors, and how it can be applied between two similar colors or in painting to set mood. Plates for demonstrating different color wheels and schemes are outlined for a final submission. Key sources are cited.
Ксения Стернина | (Mail.Ru Group)Gamer Experience Research на различных этапа...RIF-Technology
На примерах реальных кейсов Mail.Ru Group я расскажу о том, как методы UX исследований помогают разрабатывать игры на различных этапах от идеи до проверки готовых решений. Обсудим, какие методики UXR можно оставить без изменения, а какие требуют адаптации.
Pertussis, or whooping cough, is a highly contagious respiratory disease caused by the bacterium Bordetella pertussis. It is characterized by severe coughing fits that can end in a "whooping" sound when breathing in. The disease occurs in three stages - catarrhal, paroxysmal, and convalescent. It is most common and severe in infants under 6 months old. Treatment involves antibiotics and isolation. The disease is controlled through active immunization programs using DPT or DTaP vaccines, as well as prophylactic antibiotics for contacts of cases.
На примере реальных проектов и сценариев рассмотрим использование Xamarin.Forms, а также особенностей этой платформы для опытных.NET и iOS-/Android-разработчиков. Дополнительно рассмотрим интеграцию сервисов Bitrise, Xamarin Test Cloud и HockeyApp в процесс разработки.
The grid method is a technique for enlarging or reducing images accurately by overlaying a grid on the original and copied images. It involves marking equal intervals along the edges of the original image to create a grid of squares. The same grid is then drawn on the paper for the new image size. Numbers are assigned to each square. Details are then copied square by square from the original to the new image to scale it up or down accurately. This technique allows images to be enlarged or reduced to different sizes while maintaining correct proportions.
Este documento presenta dos ejemplos de algoritmos: uno para hacer un omelet y otro para poner una carga en el lavarropas. Explica las estructuras principales de los algoritmos como la secuencia de pasos y la decisión. Solicita crear algoritmos para una comida y una tarea diaria usando estas estructuras y marcando dónde se usan.
TNL College Tour '05 documents a college tour trip made by TNL to Atlanta, Georgia. The tour visited important sites like the MLK Center and various college campuses. Members of TNL expressed their commitment to continuing their education and bettering themselves, as well as gratitude to those who supported their journey. The tour was a success and provided inspiration and knowledge about moving forward with their education.
Flat Panel Digital Fluoroscopy by Akash Das AkashDas169
This document discusses flat panel digital fluoroscopy. It begins by explaining that fluoroscopy is a technique that uses a fluoroscope to obtain real-time moving images of a patient's internal structures. It then classifies fluoroscopy into C-arm and under/over table units, as well as conventional and digital units. The document goes on to explain that flat panel detectors have replaced image intensifier tubes and CCD cameras in digital fluoroscopy. Flat panel detectors consist of an array of individual detector elements and use a CsI phosphor, amorphous silicon, and thin film transistors to convert X-rays into light and then electronic signals. In closing, the mechanisms of flat panel detectors and traditional image intensifiers are compared through diagrams
Gel documentation systems are used to image and document nucleic acid and protein gels stained with ethidium bromide. They typically consist of a UV light source, hood, and camera. Ethidium bromide fluorescence allows visualization of DNA/RNA bands under UV light. Larger amounts of nucleic acid bind more ethidium bromide and appear brighter. Gel doc systems are used to capture and analyze gel images for applications like electrophoresis, blotting, and colony counting. Components include the camera, lenses, illuminator, stage, and software for image processing and analysis.
Chromatography is a technique used to separate mixtures by differential distribution between a stationary and mobile phase. It has various applications in science and medicine. The document discusses the history of chromatography, types including thin layer chromatography, gas chromatography, high performance liquid chromatography, and modes such as adsorption, partition, ion exchange, and size exclusion chromatography. Chromatography is commonly used for analytical and preparative purposes in various fields.
The document summarizes the key components and parameters of fluoroscopy systems. It discusses the image intensifier, which converts x-ray photons into light photons and uses electrodes to focus electrons onto an output screen. Parameters like conversion coefficient, brightness uniformity, and spatial resolution are described. It also covers the image intensifier's connection to a TV system using cameras like vidicons or CCDs, and how this produces a video signal to display fluoroscopy images on a monitor in real-time.
This document discusses different types of radiography detectors, focusing on radiographic film. It describes the components and construction of radiographic film, including the base, emulsion, and silver halide crystals. It explains how x-rays or light from intensifying screens interact with the emulsion to create a latent image. Key factors for selecting radiographic film types are also summarized, such as contrast, speed, resolution, crossover effects, spectral matching of screens and film, and safelighting considerations.
Computed radiography (CR) uses reusable imaging plates and associated hardware and software to acquire and display digital x-ray images as an alternative to traditional film-based radiography. The document provides an overview of the key components of a CR system, including the imaging plate, reader/digitizer, and workstation. It describes how a latent image is captured and stored in the phosphor plate from x-ray exposure, then stimulated and converted to a digital image by the reader using a laser. The advantages of CR over conventional radiography are also summarized, such as reusability of plates and improved image manipulation, storage and sharing capabilities.
Pharmaceutical imaging techniques provide visual representations of objects like body parts or pharmaceutical products for quality control, data collection, or disease diagnosis using computerized methods like ultrasound or spectroscopy. Imaging technologies are gaining attention in the pharmaceutical industry due to their potential to accelerate drug discovery and development. Common techniques discussed in the document include chemical imaging, elemental imaging, digital imaging, fluorescence correlation spectroscopy, and micro-X-ray fluorescence, which are used for applications like content uniformity testing, particle characterization, counterfeit detection, and detecting drug distribution.
This document discusses various concepts related to radiographic image quality and measurements. It defines terms like radiographic contrast, spatial resolution, contrast resolution, noise, and artifacts. It describes how factors like the film, geometry, and subject can impact radiographic quality. It also discusses optical density, sensitometry, and how the characteristic curve relates exposure to density. The modulation transfer function and how it relates to spatial frequencies is explained. Overall, the document provides an overview of key technical factors and measurements that influence the quality of radiographic images.
In vivo confocal microscopy allows for high-resolution imaging of the living cornea. It works by scanning a small region of the cornea with light to build up an image point-by-point, achieving high magnification without disrupting corneal tissue. There are three main types: tandem scanning uses a Nipkow disk, scanning slit uses a slit aperture, and laser scanning confocal microscopy is commonly used to serially image thin corneal layers. Confocal microscopy can be used to diagnose and monitor conditions like infections, dystrophies, and thinning disorders by visualizing corneal cells, structures, deposits and infiltrates. It provides rapid diagnosis and guidance for treatment.
Dharmasekara is an energetic and knowledgeable professional with a Bachelor's degree in Finance. He has over 5 years of experience working as an Audit Supervisor at KPMG Sri Lanka, where he has provided assurance, audit, and consulting services to clients in various industries. He is proficient in English, Sinhala, accounting standards, and computer programs. Dharmasekara seeks new opportunities to utilize his skills in finance, auditing, and client services.
This document defines color and describes three key properties: hue, intensity, and value. It introduces the color wheel and primary, secondary, and tertiary colors. Different color schemes are explained including complementary, analogous, triadic, split-complementary, rectangle, and square. Color temperature is defined as describing the overall warmth or coolness of colors, and how it can be applied between two similar colors or in painting to set mood. Plates for demonstrating different color wheels and schemes are outlined for a final submission. Key sources are cited.
Ксения Стернина | (Mail.Ru Group)Gamer Experience Research на различных этапа...RIF-Technology
На примерах реальных кейсов Mail.Ru Group я расскажу о том, как методы UX исследований помогают разрабатывать игры на различных этапах от идеи до проверки готовых решений. Обсудим, какие методики UXR можно оставить без изменения, а какие требуют адаптации.
Pertussis, or whooping cough, is a highly contagious respiratory disease caused by the bacterium Bordetella pertussis. It is characterized by severe coughing fits that can end in a "whooping" sound when breathing in. The disease occurs in three stages - catarrhal, paroxysmal, and convalescent. It is most common and severe in infants under 6 months old. Treatment involves antibiotics and isolation. The disease is controlled through active immunization programs using DPT or DTaP vaccines, as well as prophylactic antibiotics for contacts of cases.
На примере реальных проектов и сценариев рассмотрим использование Xamarin.Forms, а также особенностей этой платформы для опытных.NET и iOS-/Android-разработчиков. Дополнительно рассмотрим интеграцию сервисов Bitrise, Xamarin Test Cloud и HockeyApp в процесс разработки.
The grid method is a technique for enlarging or reducing images accurately by overlaying a grid on the original and copied images. It involves marking equal intervals along the edges of the original image to create a grid of squares. The same grid is then drawn on the paper for the new image size. Numbers are assigned to each square. Details are then copied square by square from the original to the new image to scale it up or down accurately. This technique allows images to be enlarged or reduced to different sizes while maintaining correct proportions.
Este documento presenta dos ejemplos de algoritmos: uno para hacer un omelet y otro para poner una carga en el lavarropas. Explica las estructuras principales de los algoritmos como la secuencia de pasos y la decisión. Solicita crear algoritmos para una comida y una tarea diaria usando estas estructuras y marcando dónde se usan.
TNL College Tour '05 documents a college tour trip made by TNL to Atlanta, Georgia. The tour visited important sites like the MLK Center and various college campuses. Members of TNL expressed their commitment to continuing their education and bettering themselves, as well as gratitude to those who supported their journey. The tour was a success and provided inspiration and knowledge about moving forward with their education.
The document celebrates successfully finishing or achieving a goal, with many repetitions of the letter O and an exclamation of "GOOOOOOOOOOOL Y FIN !!!!", indicating the goal has been reached and completed.
El documento describe los eventos de la semana del 19 al 25 de mayo de 1810 en Buenos Aires que condujeron a la formación de la Primera Junta de Gobierno. Líderes criollos presionaron al virrey Cisneros para convocar un cabildo abierto donde se votó para removerlo del poder. Tras intensos debates, el 22 de mayo el cabildo abierto votó para que Cisneros cese en su mando y el poder recaiga temporalmente en el cabildo hasta la formación de una junta de gobierno. El 24 de mayo Cisner
TNL College Tour '05 documents a college tour trip made by TNL to Atlanta, Georgia. The tour visited important sites like the MLK Center and various college campuses. Members of TNL expressed their motivation to continue moving forward and reaching higher levels of success. They were thankful for the opportunity and grateful to those who helped make the tour possible.
Live Strong Technical Services provides various fit out, maintenance, and decoration services for commercial and residential clients in the UAE. They have a team of over 15 years of combined experience in customer service, decorating, repairs, and property maintenance. Their services include air conditioning, electrical work, masonry, painting, plumbing, and more. Notable past clients include Lotus Hotels, Savoy Hotels, Le Meridien, and Kempinski hotels.
A nice way to say "Merry Christmas"! Today LTF Labortechnik received a Christmas Parcel from our partner R-Biopharm. Inside we found a testkit for the analysis of our Christmas readiness along with a humoristic instruction manual which is very much worth reading ;-) Thanks for this gorgeous surprise!
In dieser Präsentation zeigen wir Ihnen Alles, was Sie zu unseren Geldokumentations-Systemen der Firma Syngene wissen müssen. Sprechen Sie uns an unter info@labortechnik.com oder besuchen Sie uns auf unserer Website https://www.labortechnik.com/de .
Se hace un análisis de los modelos pedagógicos en el aula y la virtualidad; asímismo se habla sobre el diseño tecnopedagógico e instruccional y como se integran a los modelos pedagógicos.
Flow cytometry uses lasers to detect intrinsic cell properties like size and granularity from light scatter, and extrinsic properties from fluorescent dyes and antibodies. A flow cytometer passes single cells through a flow cell where they are interrogated by lasers. Detected light is measured and cells are analyzed individually. Good panel design considers fluorophore brightness and spectral overlap. Compensation or spectral unmixing is used to correct for signal overlap between fluorophores. Data analysis involves gating populations of interest and removing doublets or dead cells. Resources for further information are provided.
The document is a 17 page paper on flow cytometry. It begins with an introduction and overview of flow cytometry, including its basic concepts, parameters measured, and how fluorescence is involved. It then reviews a research paper on microfluidic flow cytometry, summarizing the paper's introduction, theory, design of experiment, fabrication, results, and conclusions. The paper achieved over 94% accuracy compared to flow cytometry in determining the percentages of CD4+ and CD8+ T cell subpopulations in mixed samples. It demonstrates the potential for a microchip-based system to perform label-free immune cell analysis in real-time. In the future, the measurement throughput could be improved by revising the fluidic design to lower hydraulic resistance
Immunofluorescence and fluoroscence microscopyManjubala Us
This document provides information about immunofluorescence and fluorescence microscopy techniques. It discusses immunofluorescence, which uses fluorescent-labeled antibodies to detect target antigens. It describes direct and indirect immunofluorescence methods. It also discusses fluorescence microscopy, including different types of fluorescent dyes, fluorescence microscopes, applications such as visualizing viral plaques and detecting proteins in cells, and considerations for effective immunofluorescence applications. Flow cytometry is also summarized, which uses fluorescence to examine particles like cells that are passed through a flow chamber.
dokumen.tips_immunofluorescence-and-fluoroscence-microscopy.pdfBassem Ahmed
Immunofluorescence is a technique that uses fluorescent-labeled antibodies to detect specific target antigens in cells or tissues. It allows visualization of the target under a fluorescence microscope. There are two main methods - direct immunofluorescence, which uses pre-labeled antibodies, and indirect immunofluorescence, which uses a secondary antibody labeled with a fluorophore. Immunofluorescence is widely used in research and clinical diagnosis to study the distribution of proteins, glycoproteins and other molecules in cells and tissues.
Flow cytometry is a laser-based technology used to detect and measure physical and chemical characteristics of cells or particles. It allows for rapid analysis of multiple characteristics of cells, including size, granularity, and fluorescence intensity. The key components of a flow cytometer include lasers, optical filters, detectors, and fluidics and optics systems to analyze cells in suspension. As cells pass through the laser beam, light is scattered or absorbed, detected, and converted to digital signals for analysis. Applications include cell sorting, cell cycle analysis, and clinical diagnostics. Detection of apoptosis can be done based on changes in light scattering, membrane asymmetry detected by annexin V binding, and other markers.
Flow cytometry definition, principle, parts, steps, types, usesGayathri Devi S
Flow cytometry is a technique that uses lasers to detect and measure physical and chemical characteristics of cells or particles in fluid suspension. Cells pass through a laser beam, which scatters light and causes fluorescence that is detected by sensors. Measurements of scattered and fluorescent light provide information about cell size, granularity, and expression of targeted proteins or nucleic acids. Flow cytometry allows rapid multi-parameter analysis of individual cells in heterogeneous populations and is widely used for clinical, research, and industrial applications.
This document discusses spectrophotometry and the Beer-Lambert law. It provides:
1) An overview of how spectrophotometers work by measuring the absorption of light by chemical compounds and relating absorption to concentration according to the Beer-Lambert law.
2) A description of the basic components of a spectrophotometer including a light source, wavelength selector, sample cuvette, detector, and readout device.
3) Explanations of how to prepare and measure standards to generate a calibration curve to determine unknown concentrations.
This document provides information about using a spectrophotometer for quantitative analysis. It discusses how spectrophotometers work based on the Beer-Lambert law relating absorbance of light to analyte concentration. The key components of a spectrophotometer are described including the light source, wavelength selector, sample cuvette, detector, and readout device. General procedures are outlined for preparing standard solutions to generate a calibration curve and determining concentrations of unknown samples.
This document provides information about using a spectrophotometer for quantitative analysis. It discusses how spectrophotometers work based on the Beer-Lambert law relating absorbance of light to analyte concentration. The key components of a spectrophotometer are described including the light source, wavelength selector, sample cuvette, detector, and readout device. General procedures are outlined for preparing standard solutions to generate a calibration curve and determining concentrations of unknown samples.
Flow cytometry allows for the quantitative and qualitative analysis of cell populations by measuring physical and chemical characteristics of individual cells as they flow in a single file through a laser beam. Cells are labeled with fluorescent markers and passed through the flow cytometer which measures light scattering and fluorescence emission. Data is collected and analyzed using plots that display fluorescence parameters of individual cells. Flow cytometry has many clinical applications including immunophenotyping, stem cell analysis, and detection of rare cell populations in diseases like cancer.
Using the 3D Cell Explorer-fluo for fluorescence and holotomographic imagingMathieuFRECHIN
Nanolive’s 3D Cell Explorer allows for the creation of very powerful 3D images and 4D time lapses
of living cells with very high spatio-temporal resolution (x,y:180nm; z:400nm; t:1.7sec). Moreover,
imaging with the 3D Cell Explorer does not require the use of any labels since the microscope directly
measures the refractive index of the different substructures of the cell. However, in the context of
molecular or cellular biology investigations, it can be useful to follow fluorescent markers combined
with the refractive index distribution to validate specific structures or to correlate fluorescent signals
and cellular states.
For this purpose, Nanolive developed the 3D Cell Explorer-fluo (https://nanolive.ch/fluo/) – a
complete solution that combines high precision tomographic data with high quality tri- or fourchannel
epifluorescence provided by a CoolLed module (https://www.coolled.com/).
In this application note we will present the time lapse imaging of mouse embryonic stem cells
(mESCs) that have been genetically modified to express the fluorescence ubiquitination cell cycle
indicator (FUCCI), a two-color (red and green) indicator that allows to monitor the cell cycle phases.
We will explain how to use the 3D Cell Explorer-fluo to record movies that require both fluorescence
and 3D refractive index imaging and will propose a solution to analyze the resulting time lapse
experiment.
The document summarizes the development and validation of a hyperspectral imaging microscopy system called Xanoscope for pathology applications. Key points:
1) Xanoscope allows for fast, automated acquisition of hyperspectral images over contiguous wavelength bands with high spatial resolution, reducing scan times significantly compared to previous methods.
2) Validation experiments showed Xanoscope produces quantitative measurements and improves signal-to-noise ratio through optimized camera settings and image accumulation.
3) Xanoscope was used to scan 10 multiplexed fluorophores in cell lines, and linear unmixing was able to measure the individual contribution of each fluorophore at pixel-level for advanced pathological analysis.
Review on laser scanning confocal microscopyUdayan Ghosh
This document provides an overview of laser scanning confocal microscopy (LSCM). It discusses the working principle of LSCM, highlighting how it uses pinholes and laser excitation to optically section samples. Advantages include high resolution 3D imaging without physical sectioning. Applications described include biology, materials science, and semiconductor fabrication. LSCM is presented as a valuable non-destructive characterization tool across many fields due to its high resolution and ability to image living samples.
Derivative spectroscopy involves converting a normal UV-Vis absorption spectrum into its first or second derivative spectrum. This allows removal of spectral interferences and increases selectivity for analytical determinations. Derivative spectra are generated mathematically or by using a dual-beam spectrophotometer with a small wavelength interval between the beams. This provides better resolution of overlapping bands and permits more accurate determination of components in multi-component mixtures compared to conventional absorption spectroscopy. Derivative spectroscopy finds applications in pharmaceutical analysis and other areas requiring determination of individual components in the presence of interferants.
The document provides an overview of the basic principles and components of flow cytometry. It discusses how flow cytometry works by measuring the properties of cells in fluid flow, using a combination of fluidics to introduce cells, optics to generate and collect light signals, and electronics to convert signals to digital data. Key aspects summarized include how cells are hydrodynamically focused and interrogated by light scatter and fluorescence to derive information on their size, granularity, and marker expression that can be analyzed using software.
The document discusses small animal imaging techniques. It provides an overview of various imaging modalities including their resolutions and applications. Specific techniques covered include bioluminescence imaging, fluorescence imaging, computed tomography, magnetic resonance imaging, positron emission tomography, and single photon emission computed tomography. The document also discusses fluorescent probes and reagents used for small animal imaging including near-infrared dyes, quantum dots, fluorescent microspheres, and protease-activatable probes. Potential applications highlighted include tumor detection, vascular imaging, and monitoring of enzyme activity.
The document describes several analytical instrumentation facilities available at Virginia Commonwealth University's Department of Chemistry, including mass spectrometry, NMR spectroscopy, and ICP-MS. It provides details on specific instruments and techniques available at each facility, such as FT-IR spectrometers, Raman spectrometers, GC/MS, and fluorescence spectrophotometers. It also outlines training procedures and requirements to use the NMR facility.
PCR is a technique for amplifying DNA sequences. It requires template DNA, reaction buffer, magnesium ions, dNTPs, primers, and DNA polymerase. Variations include colony PCR, nested PCR, and real-time PCR, which uses fluorescent probes to detect amplification in real time. Common probe types are SYBR Green dyes, TaqMan probes, molecular beacons, and hybridization probes, which use FRET between donor and acceptor dyes. Real-time PCR instruments contain excitation sources and fluorometers to detect fluorescence levels during thermal cycling.
Similar to Geldokumentations-Systeme von Syngene: ein Anwendungsleitfaden (20)
LTF Labortechnik hat heute diesen süßen Weihnachtsgruß von seinem Partner R-Biopharm erhalten. Der RIDASDREEN Weihnachtstestkit mit spezieller, humorvoller Bedienungsanleitung testet die Weihnachtsstimmung Ihrer Mitarbeiter und hinterlässt ein garantiert gutes Gefühl!
Diese Übersicht verdeutlicht das intelligente Design unserer Laborautomatisierungs-Lösung PIRO. Ein Pipettierroboter, der keine Wünsche offen lässt! Sprechen Sie uns an unter info@labortechnik.com oder besuchen Sie uns auf unserer Website https://www.labortechnik.com/de .
The document summarizes the MyGo Mini real-time PCR instrument. It is the world's most compact PCR instrument with no moving parts, enabling silent operation. It provides high precision and analytical sensitivity, able to discriminate samples that differ by as little as 10% gene expression. The solid-state design makes it highly reliable with optional 5-year warranty. It has easy-to-use software for automated analysis and reporting of PCR and high-resolution melt data.
Diese Broschüre beschreibt die Einzigartigkeit dieses innovativen und perfekt designten qPCR Realtime-Cyclers. Welcher qPCR-Cycler bietet sonst noch 120 optische Kanäle, die gleichzeitige Abarbeitung von TaqMan und Hybridisierungssonden, 7plex-Analysen, und das Alles bei einer thermischen Uniformität von 0,05°C? Sprechen Sie uns an unter info@labortechnik.com oder besuchen Sie uns auf unserer Website https://www.labortechnik.com/de .
In dieser Präsentation wird gezeigt, wie ein bekannter deutscher qPCR Testkithersteller seine Testkits auf unserem MyGo Pro Realtime PCR-Cycler getestet und optimiert hat. Sprechen Sie uns an unter info@labortechnik.com oder besuchen Sie uns auf unserer Website https://www.labortechnik.com/de .
In dieser Anleitung beschreiben wir Schritt für Schritt, wie Sie Ihre Gele optimal auswerten. Beschrieben ist auch die Vorbereitung und der Export für eine Publikation. Sprechen Sie uns an unter info@labortechnik.com oder besuchen Sie uns auf unserer Website https://www.labortechnik.com/de .
The ability to recreate computational results with minimal effort and actionable metrics provides a solid foundation for scientific research and software development. When people can replicate an analysis at the touch of a button using open-source software, open data, and methods to assess and compare proposals, it significantly eases verification of results, engagement with a diverse range of contributors, and progress. However, we have yet to fully achieve this; there are still many sociotechnical frictions.
Inspired by David Donoho's vision, this talk aims to revisit the three crucial pillars of frictionless reproducibility (data sharing, code sharing, and competitive challenges) with the perspective of deep software variability.
Our observation is that multiple layers — hardware, operating systems, third-party libraries, software versions, input data, compile-time options, and parameters — are subject to variability that exacerbates frictions but is also essential for achieving robust, generalizable results and fostering innovation. I will first review the literature, providing evidence of how the complex variability interactions across these layers affect qualitative and quantitative software properties, thereby complicating the reproduction and replication of scientific studies in various fields.
I will then present some software engineering and AI techniques that can support the strategic exploration of variability spaces. These include the use of abstractions and models (e.g., feature models), sampling strategies (e.g., uniform, random), cost-effective measurements (e.g., incremental build of software configurations), and dimensionality reduction methods (e.g., transfer learning, feature selection, software debloating).
I will finally argue that deep variability is both the problem and solution of frictionless reproducibility, calling the software science community to develop new methods and tools to manage variability and foster reproducibility in software systems.
Exposé invité Journées Nationales du GDR GPL 2024
Nucleophilic Addition of carbonyl compounds.pptxSSR02
Nucleophilic addition is the most important reaction of carbonyls. Not just aldehydes and ketones, but also carboxylic acid derivatives in general.
Carbonyls undergo addition reactions with a large range of nucleophiles.
Comparing the relative basicity of the nucleophile and the product is extremely helpful in determining how reversible the addition reaction is. Reactions with Grignards and hydrides are irreversible. Reactions with weak bases like halides and carboxylates generally don’t happen.
Electronic effects (inductive effects, electron donation) have a large impact on reactivity.
Large groups adjacent to the carbonyl will slow the rate of reaction.
Neutral nucleophiles can also add to carbonyls, although their additions are generally slower and more reversible. Acid catalysis is sometimes employed to increase the rate of addition.
The use of Nauplii and metanauplii artemia in aquaculture (brine shrimp).pptxMAGOTI ERNEST
Although Artemia has been known to man for centuries, its use as a food for the culture of larval organisms apparently began only in the 1930s, when several investigators found that it made an excellent food for newly hatched fish larvae (Litvinenko et al., 2023). As aquaculture developed in the 1960s and ‘70s, the use of Artemia also became more widespread, due both to its convenience and to its nutritional value for larval organisms (Arenas-Pardo et al., 2024). The fact that Artemia dormant cysts can be stored for long periods in cans, and then used as an off-the-shelf food requiring only 24 h of incubation makes them the most convenient, least labor-intensive, live food available for aquaculture (Sorgeloos & Roubach, 2021). The nutritional value of Artemia, especially for marine organisms, is not constant, but varies both geographically and temporally. During the last decade, however, both the causes of Artemia nutritional variability and methods to improve poorquality Artemia have been identified (Loufi et al., 2024).
Brine shrimp (Artemia spp.) are used in marine aquaculture worldwide. Annually, more than 2,000 metric tons of dry cysts are used for cultivation of fish, crustacean, and shellfish larva. Brine shrimp are important to aquaculture because newly hatched brine shrimp nauplii (larvae) provide a food source for many fish fry (Mozanzadeh et al., 2021). Culture and harvesting of brine shrimp eggs represents another aspect of the aquaculture industry. Nauplii and metanauplii of Artemia, commonly known as brine shrimp, play a crucial role in aquaculture due to their nutritional value and suitability as live feed for many aquatic species, particularly in larval stages (Sorgeloos & Roubach, 2021).
Deep Behavioral Phenotyping in Systems Neuroscience for Functional Atlasing a...Ana Luísa Pinho
Functional Magnetic Resonance Imaging (fMRI) provides means to characterize brain activations in response to behavior. However, cognitive neuroscience has been limited to group-level effects referring to the performance of specific tasks. To obtain the functional profile of elementary cognitive mechanisms, the combination of brain responses to many tasks is required. Yet, to date, both structural atlases and parcellation-based activations do not fully account for cognitive function and still present several limitations. Further, they do not adapt overall to individual characteristics. In this talk, I will give an account of deep-behavioral phenotyping strategies, namely data-driven methods in large task-fMRI datasets, to optimize functional brain-data collection and improve inference of effects-of-interest related to mental processes. Key to this approach is the employment of fast multi-functional paradigms rich on features that can be well parametrized and, consequently, facilitate the creation of psycho-physiological constructs to be modelled with imaging data. Particular emphasis will be given to music stimuli when studying high-order cognitive mechanisms, due to their ecological nature and quality to enable complex behavior compounded by discrete entities. I will also discuss how deep-behavioral phenotyping and individualized models applied to neuroimaging data can better account for the subject-specific organization of domain-general cognitive systems in the human brain. Finally, the accumulation of functional brain signatures brings the possibility to clarify relationships among tasks and create a univocal link between brain systems and mental functions through: (1) the development of ontologies proposing an organization of cognitive processes; and (2) brain-network taxonomies describing functional specialization. To this end, tools to improve commensurability in cognitive science are necessary, such as public repositories, ontology-based platforms and automated meta-analysis tools. I will thus discuss some brain-atlasing resources currently under development, and their applicability in cognitive as well as clinical neuroscience.
Unlocking the mysteries of reproduction: Exploring fecundity and gonadosomati...AbdullaAlAsif1
The pygmy halfbeak Dermogenys colletei, is known for its viviparous nature, this presents an intriguing case of relatively low fecundity, raising questions about potential compensatory reproductive strategies employed by this species. Our study delves into the examination of fecundity and the Gonadosomatic Index (GSI) in the Pygmy Halfbeak, D. colletei (Meisner, 2001), an intriguing viviparous fish indigenous to Sarawak, Borneo. We hypothesize that the Pygmy halfbeak, D. colletei, may exhibit unique reproductive adaptations to offset its low fecundity, thus enhancing its survival and fitness. To address this, we conducted a comprehensive study utilizing 28 mature female specimens of D. colletei, carefully measuring fecundity and GSI to shed light on the reproductive adaptations of this species. Our findings reveal that D. colletei indeed exhibits low fecundity, with a mean of 16.76 ± 2.01, and a mean GSI of 12.83 ± 1.27, providing crucial insights into the reproductive mechanisms at play in this species. These results underscore the existence of unique reproductive strategies in D. colletei, enabling its adaptation and persistence in Borneo's diverse aquatic ecosystems, and call for further ecological research to elucidate these mechanisms. This study lends to a better understanding of viviparous fish in Borneo and contributes to the broader field of aquatic ecology, enhancing our knowledge of species adaptations to unique ecological challenges.
Travis Hills' Endeavors in Minnesota: Fostering Environmental and Economic Pr...Travis Hills MN
Travis Hills of Minnesota developed a method to convert waste into high-value dry fertilizer, significantly enriching soil quality. By providing farmers with a valuable resource derived from waste, Travis Hills helps enhance farm profitability while promoting environmental stewardship. Travis Hills' sustainable practices lead to cost savings and increased revenue for farmers by improving resource efficiency and reducing waste.
Or: Beyond linear.
Abstract: Equivariant neural networks are neural networks that incorporate symmetries. The nonlinear activation functions in these networks result in interesting nonlinear equivariant maps between simple representations, and motivate the key player of this talk: piecewise linear representation theory.
Disclaimer: No one is perfect, so please mind that there might be mistakes and typos.
dtubbenhauer@gmail.com
Corrected slides: dtubbenhauer.com/talks.html
ANAMOLOUS SECONDARY GROWTH IN DICOT ROOTS.pptxRASHMI M G
Abnormal or anomalous secondary growth in plants. It defines secondary growth as an increase in plant girth due to vascular cambium or cork cambium. Anomalous secondary growth does not follow the normal pattern of a single vascular cambium producing xylem internally and phloem externally.
Remote Sensing and Computational, Evolutionary, Supercomputing, and Intellige...University of Maribor
Slides from talk:
Aleš Zamuda: Remote Sensing and Computational, Evolutionary, Supercomputing, and Intelligent Systems.
11th International Conference on Electrical, Electronics and Computer Engineering (IcETRAN), Niš, 3-6 June 2024
Inter-Society Networking Panel GRSS/MTT-S/CIS Panel Session: Promoting Connection and Cooperation
https://www.etran.rs/2024/en/home-english/
BREEDING METHODS FOR DISEASE RESISTANCE.pptxRASHMI M G
Plant breeding for disease resistance is a strategy to reduce crop losses caused by disease. Plants have an innate immune system that allows them to recognize pathogens and provide resistance. However, breeding for long-lasting resistance often involves combining multiple resistance genes
2. Syngene Applications Training Guide 2
Contents Page
Lighting and Filter combinations………………………………………………………………
Examples…………………………………………………………………………………………..
Chemiluminescence versus Fluorescence Western blot detection…………….
Film versus digital imaging Chemiluminescence Western blot………………….
Dot Blot Protocol…………………………………………………………………………………….
Fluorescent Western blot general protocol………………………………………………
General Tips and advice for Fluorescence Western blots………………………….
General Tips and advice for IR Western blots…………………………………………..
3
6
8
9
10
11
13
15
3. Syngene Applications Training Guide 3
Lighting and Filter combinations
Fluorescence
Excitation
When a photon of energy supplied by an external source such as a LED or a laser is absorbed by a
fluorophore it creates an excited unstable electronic singlet state (when an electron in a molecule
with a singlet ground state is excited to a higher energy level).
Emission
When a fluorochrome molecule falls from the excited state to the ground state light is often emitted
at a characteristic wavelength.
Excitation and Emission spectra
Excitation Spectra
This spectrum is a plot of emitted fluorescence versus excitation wavelength.
Figure 1- Jablonski diagram illustrating the processes
involved in creating an excited electronic singlet state by
optical absorption and subsequent emission of
fluorescence. 1) Excitation; 2) vibrational relaxation; 3)
Emission
4. Syngene Applications Training Guide 4
Emission spectra
A plot of relative intensity of emitted light as a function of the emission wavelength.
Excitation sources
Light sources fall into two broad categories—wide-area, broad-wavelength sources, such as UV and
xenon arc lamps, and line sources with discrete wavelengths, such as lasers. Broad wavelength
excitation sources are used in fluorescence camera imaging systems. The spectral output of a lamp is
broad, but it can be tuned to a narrow band of excitation light with the use of filters.
Excitation light is delivered to the sample by direct illumination of the imaging field, with the
excitation source positioned either above or below the sample.
Filter Selection
Matching a fluorochrome label with a suitable excitation source and emission filter is the key to
optimal detection efficiency.
There are three main types of filters;
Long-pass (LP) filters pass light that is longer than a specified wavelength and reject all shorter
wavelengths. A good quality long-pass filter is characterized by a steep transition between rejected
and transmitted wavelengths. Long-pass filters are named for the wavelength at the midpoint of the
transition between the rejected and transmitted light (cutoff point).
Short-pass (SP) filters reject wavelengths that are longer than a specified value and pass shorter
wavelengths. Like long-pass filters, short-pass filters are named according to their cutoff point.
Band-pass (BP) filters allow a band of selected wavelengths to pass through, while rejecting all
shorter and longer wavelengths. Band-pass filters provide very sharp cutoffs with very little
transmission of the rejected wavelengths.
The full-width at half-maximum transmission (FWHM). Band-pass filters with an FWHM of 20–30 nm
are optimal for most fluorescence applications, including multi-label experiments.
5. Syngene Applications Training Guide 5
Figure 2- Transmission profile for a band-pass
(670 BP30) filter. The full-width at half maximum
(FWHM) transmission of 30nm is indicated by the
arrows.
8. Syngene Applications Training Guide 8
Chemiluminescence versus Fluorescence Western blot detection
The current trend with many researchers is the move towards changing their Western blot detection
method from chemiluminescence to fluorescence and fluorescence multiplexing.
There are several reasons for this trend the main reason is that fluorescent detection allows the user
to multiplex their Western blots which allows simultaneous detection of several target proteins at
once reducing or eliminating the need to strip and re-probe the membrane which can lead to a loss
of sensitivity and is time-consuming.
The following table outlines the advantages of using fluorescence over chemiluminescence.
Feature Chemiluminescence Fluorescence
Detection Single-plex reaction cannot
distinguish light from light
Multiplex detection can detect
more than 2 fluorophores
simultaneously
Quantification Cannot quantify target protein
compared to internal control
(housekeeping gene) at the same
time.
Film has a relatively restricted
linear dynamic range.
Easy quantification by comparing
one protein’s abundance to an
internal control (housekeeping
gene).
Relatively wide linear dynamic
range up to 3 orders of magnitude.
Reproducibility Small differences during set-up
e.g. timing, freshness of reagents
can change the total light signal
output. Enzymatic reaction.
Fluorescence is fairly constant.
Given fluorophore will produce a
specific amount of light no more
no less. Stable.
Sensitivity Good sensitivity depending on
which chemi kit is used. Maybe
better for very low abundant
protein detection.
Sensitive. Better sensitivity in red
and IR wavelengths.
Stability Depends on chemiluminescent kit
used but typically stable for no
more than 24 hours.
Can image only once for optimum
results.
Stable for several months or
longer. Can be re-imaged several
times without significant loss of
signal.
9. Syngene Applications Training Guide 9
Film versus digital imaging Chemiluminescence Western blot
The question of whether digital imaging is more sensitive than film has been greatly debated over
the years. With the advancement of CCD imaging systems more researchers are moving away from
exposing their blots to film.
When assessing the performance of both methods the most important criteria to look at are
dynamic range and the limit of detection (LOD).
A digital imager typically records from 4,096 to 65,536 different levels of intensity, thus covering a
greater dynamic range of grey scale. Thus, the dynamic range digital imagers are on average 2-4
orders of magnitude higher than film. This however is theoretical and in practice the dynamic range
is determined from running a dilution series of protein (e.g 10, 5, 2.5ng) to determine the LOD. The
following equation is then used
Max (concentration protein) = X then LOG10 (X) = dynamic range
Min (concentration protein)
The majority of chemiluminescent kits provide a dynamic range of about 2 orders of magnitude with
an imaging system.
One of film’s major limitations is its limited linear dynamic range for light detection; film is easily
saturated by chemiluminescent signals from the blot. It’s this ease of signal saturation that gives
scientists the idea that film is more sensitive than digital imaging.
Film has a higher LOD compared to that of digital imaging systems and therefore, weak signals need
extensive exposure time to be detected and quantified on film. Digital imaging systems will capture
less intense signals that are missed by film and do so without compromising stronger signals to
saturation.
The following table highlights the advantages of using an imaging system over film.
Feature Film Digital Imaging system
Dynamic Range Limited linear dynamic range
difficult to see faint and bright
bands without over saturation
Much broader dynamic range
can detect bright and faint
bands without causing
saturation
Linearity Non-linear Large linear response
Cost Expensive maintaining a
darkroom, chemicals and film
Cheaper running costs only
need to invest in an imaging
system and lower operational
costs
10. Syngene Applications Training Guide 10
Dot Blot Protocol
A technique for detecting, analyzing, and identifying proteins, similar to the western blot technique but
differing in that protein samples are not separated electrophoretically but are spotted through circular
templates directly onto the membrane or paper substrate.
Concentration of proteins in crude preparations (such as culture supernatant) can be estimated
semiquantitatively by using “Dot Blot” method if you have both purified protein and specific antibody
against it.
Reagents
TBS: or PBS
20 mM Tris-HCl
150 mM NaCl
pH 7.5
TBS-T: (PBST)
0.05% Tween20 in TBS
BSA/TBS-T:
3-5% BSA in TBS-T (PSB-T)
PVDF membrane
Procedure
1. Have PVDF membrane ready, draw grid by pencil to indicate the region you are going to blot
(seebelow).
Decide what concentrations of primary and secondary antibody you wish to test and the concentration
range of protein.
2. Pipet 2μl of protein sample onto the PVDF membrane at the center of the grid. Minimize the area
that the solution penetrates (usually 3-4 mm diam.) by applying it slowly.
3. Block non-specific sites by soaking in 5% BSA in TBS-T (0.5-1 hr, RT). Use 10cm Petri Dish for
reaction chamber. Cut up the grid.
4. Incubate with primary antibody (each square being incubated with a different concentration of
primary antibody) dissolved in BSA/TBS-T for 30 min at RT.
5. Wash three times with TBS-T (3 x 5 min).
6. Incubate with secondary antibody (again different concs of the secondary antibody) for optimum
dilution, follow the manufacturer’s recommendation) for 30 min-1hr at RT.
7. Wash three times with TBS-T (15 min x 1, 5 min x 2), then once with TBS (5 min).
8. Image using the gel imager. Try several different lengths of exposure.
9. See which concentration combination works best. Gives you the most signal.
11. Syngene Applications Training Guide 11
Fluorescent Western blot general protocol
1. Perform SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transfer the protein to the
membrane (electroblotting) either by wet transfer or semi-dry transfer.
2. Wash the membrane twice with distilled water. If desired, stain the membrane with Ponceau
Red solution for 5 minutes to visualize protein bands. (Stock solution: 2% Ponceau S in 30%
trichloroacetic acid and 30% sulfosalicylic acid; dilute 1:10 for use.) Rinse the membrane in
water until protein bands are distinct then once the bands have been detected wash the
membrane in TBST to remove the Ponceau red stain. Note: Do not let the blot dry out at any
step through development, as this will cause an increase in background staining.
3. Block the blotted membrane in freshly prepared TBS and/or PBS containing BSA (3–5%) (Other
blocking reagents are available) for 30–60 minutes at room temperature with constant
agitation. A maximum blocking time of 2 hours at room temperature should not be exceeded
since staining artifacts will appear. Note: If longer blocking times are required, the membrane
should be kept at 4°C.
4. Dilute the primary antibody to the recommended concentration/dilution in fresh blocking
solution (TBS and/or PBS /3%or 5% BSA). Incubate the membrane in the primary antibody
solution for 1 to 2 hours at room temperature or overnight at 4°C with agitation.
5. Wash the membrane three times for 3 to 5 minutes each with TBS and/or PBS containing 0.05%
Tween-20. Incubate the membrane in the secondary antibody reagent of choice for 30 minutes
to 1 hour at room temperature. For example, if multiplexing two or more fluorophores requires
careful selection of primary and secondary antibodies. If two primary antibodies are derived
from different host species (e.g. primary antibodies from mouse and chicken), secondary
antibodies derived from the same host and labelled with different colour fluorophores must be
used e.g. IRDye 800CW Donkey anti-mouse and IR dye 680LT donkey anti-chicken.
Note: It is highly recommended that before combining primary antibodies in a two or more
colour experiment you should always perform preliminary blots with each primary antibody
alone to determine the expected banding pattern and possible background bands (non-
specific).
Slight cross-reactivity may occur and can complicate the interpretation of a blot, particularly if
the antigen is very abundant. If cross-reactivity is a problem, load less protein or reduce the
12. Syngene Applications Training Guide 12
amount of antibody. Always use highly cross-absorbed secondary antibodies for two colour
detection. Failure to use highly crossed absorbed antibodies may result in cross-reactivity.
6. Wash the membrane five times for 3 to 5 minutes each time with TBS and/or PBS containing
0.05%Tween-20.
Note: Tween-20 detergent has the potential to strip low affinity primary antibodies, and
therefore the membrane is briefly washed to improve the background.
7. Imaging the membrane either completely the dry the membrane or keep it wet whilst imaging. If
the membrane starts to dry out then patches can appear on the membrane which can possibly
obscure bands. Make sure that the membrane is placed on the black plastic anti-reflective
screen whilst imaging.
13. Syngene Applications Training Guide 13
General Tips and advice for Fluorescence Western blots
General Tips
1. It is important to block membranes for at least 1hr or overnight at 40
C. Be sure to
use enough blocking buffer to cover the membrane (minimum recommended is
0.4mL/cm2
). If using anti-goat antibodies a milk based blocker can interfere with
detection. They can also deteriorate rapidly at 40
C. BSA blocking buffers may cause
high membrane background.
2. Diluting antibodies- depends on the antibody and should be determined empirically.
Usually you can find a suggested starting range on the manufacturer’s data sheet.
3. Protect the membrane from light during secondary antibody incubations and washes
4. To concentrate the target protein use the narrowest well size possible for the
loading volume
5. For proteins <100KDa, try blotting in standard Tris-glycine buffer with 20% methanol
and no SDS. Addition of SDS to the transfer buffer can greatly reduce binding of
transferred proteins to the membrane.
6. It is recommended to soak the gel in transfer buffer for 10-20 minutes before setting
up transfer. Soaking the gel allows it to equilibrate the gel and removes buffer salts
that will be carried over into the transfer tank.
7. To maximise retention of transferred proteins on the membrane, allow the
membrane to completely air-dry after transfer (approx. 1-2 hours).
8. To enhance signal, try extended primary antibody incubation at room temperature
or overnight incubation at 40
C. Try and avoid extended incubations in secondary
antibodies.
Optimisation Tips
1. No single blocking reagent will be optimal for every antigen-antibody pair. Some
primary antibodies may exhibit greatly reduced signal or different non-specific
binding in different blocking solutions. It is difficult to detect target protein, changing
the blocking solution may dramatically improve performance.
2. Let the membrane dry out completely before imaging.
Two or more colour detection
Two or more colour detection requires careful selection of primary and secondary
antibodies. If two primary antibodies are derived from different host species (e.g. primary
antibodies from mouse and chicken), secondary antibodies derived from the same host and
14. Syngene Applications Training Guide 14
labelled with different colour fluorophores must be used e.g. IRDye 800CW Donkey anti-
mouse and IR dye 680LT donkey anti-chicken.
It is highly recommended that before combining primary antibodies in a two or more colour
experiment you should always perform preliminary blots with each primary antibody alone
to determine the expected banding pattern and possible background bands (non-specific).
Slight cross-reactivity may occur and can complicate the interpretation of a blot, particularly
if the antigen is very abundant. If cross-reactivity is a problem, load less protein or reduce
the amount of antibody. Always use highly cross-absorbed secondary antibodies for two
colour detection. Failure to use highly crossed absorbed antibodies may result in cross-
reactivity.
15. Syngene Applications Training Guide 15
General Tips and advice for IR Western blots
General Tips
1. It is important to block membranes for at least 1hr or overnight at 40
C. Be sure to
use enough blocking buffer to cover the membrane (minimum recommended is
0.4mL/cm2
). If using anti-goat antibodies a milk based blocker can interfere with
detection. They can also deteriorate rapidly at 40
C. BSA blocking buffers may cause
high membrane background.
2. Diluting antibodies- depends on the antibody and should be determined empirically.
Usually you can find a suggested starting range on the manufacturer’s data sheet.
3. Protect the membrane from light during secondary antibody incubations and washes
4. To concentrate the target protein use the narrowest well size possible for the
loading volume
5. For proteins <100KDa, try blotting in standard Tris-glycine buffer with 20% methanol
and no SDS. Addition of SDS to the transfer buffer can greatly reduce binding of
transferred proteins to the membrane.
6. It is recommended to soak the gel in transfer buffer for 10-20 minutes before setting
up transfer. Soaking the gel allows it to equilibrate the gel and removes buffer salts
that will be carried over into the transfer tank.
7. To maximise retention of transferred proteins on the membrane, allow the
membrane to completely air-dry after transfer (approx. 1-2 hours).
8. To enhance signal, try extended primary antibody incubation at room temperature
or overnight incubation at 40
C. Try and avoid extended incubations in secondary
antibodies.
Optimisation Tips
1. No single blocking reagent will be optimal for every antigen-antibody pair. Some
primary antibodies may exhibit greatly reduced signal or different non-specific
binding in different blocking solutions. It is difficult to detect target protein, changing
the blocking solution may dramatically improve performance.
2. Let the membrane dry out completely before imaging.
Two colour detection
Two colour detection requires careful selection of primary and secondary antibodies. If two
primary antibodies are derived from different host species (e.g. primary antibodies from
mouse and chicken), IRdye secondary antibodies derived from the same host and labelled
16. Syngene Applications Training Guide 16
with different IRDye fluorophores must be used e.g. IRDye 800CW Donkey anti-mouse and
IR dye 680LT donkey anti-chicken.
It is highly recommended that before combining primary antibodies in a two colour
experiment you should always perform preliminary blots with each primary antibody alone
to determine the expected banding pattern and possible background bands (non-specific).
Slight cross-reactivity may occur and can complicate the interpretation of a blot, particularly
if the antigen is very abundant. If cross-reactivity is a problem, load less protein or reduce
the amount of antibody. Always use highly cross-absorbed secondary antibodies for two
colour detection. Failure to use highly crossed absorbed antibodies may result in cross-
reactivity.
Imaging Tips
1. It is recommended to either image the blot wet or to let the blot completely dry out.
If the membrane starts to dry out this can create blotches on the membrane which
can prevent bands being detected easily.
2. For more even illumination place one of each colour LED module opposite each
other inside the darkroom.
3. Make sure that you zoom in on the sample to completely fill the screen.
4. If you are imaging IRDye800 you may not see any bands in the preview screen of the
image capture software which can make it difficult to make sure your sample is in
focus. Please use a business card or a piece of paper with writing on it to make sure
you have the sample in focus.
5. Always handle the membrane with gloves and tweezers.
6. Do not write in pencil or pen on the membrane as they will fluoresce and can
interfere with autocapture mode in GeneSys software.