Western blot is a commonly used method for protein analysis. It can be used for qualitative and semi-quantitative protein analysis. For the accomplishment of the western blot, there are three elements, separation of proteins by size, transferring proteins to a solid support, and marking proteins by primary and secondary antibodies for visualization.
In this slide contains principle, types, methods and application of Western Blotting Technique.
Presented by: T.NIRANJAN REDDY (Department of pharmacology).
RIPER, anantapur
gives a very brief info about western blotting procedures, attractive slides, with creative animation effects, i hope this ppt of mine works good for seminar and for educational purposes.
Assignment on General principles of ImmunoassayDeepak Kumar
Assignment on General principles of immunoassay: theoretical basis and optimization of immunoassay, heterogeneous and homogenous immunoassay systems. Immunoassay methods evaluation; protocol outline, objectives and preparation. Immunoassay for digoxin and insulin
ABSTRACT: The ELISA technique is a simple, sensitive, rapid, reliable, and versatile assay system for the quantitation of antigens and antibodies. Because of the extreme discriminating power of antibodies to recognize an almost infinite array of antigenic structures, the application of ELISA to analyte measurement is almost unlimited. ELISAs have been developed in many configurations depending on the particular application of the assay.
In solid-phase ELISA, one of the immunoreactants (antibody or antigen) is immobilized onto a solid support (microtiter plate) by adsorption, through non-covalent interactions. The immobilized antibody is then incubated with test solution containing the analyte of interest. Following a period of incubation and washing, the bound antigen is detected, by the addition of an enzyme-conjugated antibody that binds to the remaining antigenic sites on the antigen.
Although the technique is easy to perform and quite sensitive, there are certain problems to be solved before it becomes widely usable. In the present Memorandum the technical details are given and the advantages and shortcomings of the procedure are discussed. Present applications and future prospects are reviewed.
OTECHNOLOGY IS CHALLENGING SUBJECT TO TEACH AND UNDERSTAND ALSO .....THEIR INTERESTING PART IS TO LEARN ABOUT MICROBIAL GENETICS AND THEIR METHODS OF GENE TRANSFER
We are engaged in manufacturing, supplying and exporting of Laboratory Filtration Products (syringe filters, membrane filters, glass fiber filters and filter papers). These filtration products are precisely manufactured by making use of superior quality raw material and ultra-modern techniques by the experience team. Our offered products are widely used in various laboratories for various filtration works in worldwide. http://www.axivasichem.com/syringe-filters.aspx
In this slide contains principle, types, methods and application of Western Blotting Technique.
Presented by: T.NIRANJAN REDDY (Department of pharmacology).
RIPER, anantapur
gives a very brief info about western blotting procedures, attractive slides, with creative animation effects, i hope this ppt of mine works good for seminar and for educational purposes.
Assignment on General principles of ImmunoassayDeepak Kumar
Assignment on General principles of immunoassay: theoretical basis and optimization of immunoassay, heterogeneous and homogenous immunoassay systems. Immunoassay methods evaluation; protocol outline, objectives and preparation. Immunoassay for digoxin and insulin
ABSTRACT: The ELISA technique is a simple, sensitive, rapid, reliable, and versatile assay system for the quantitation of antigens and antibodies. Because of the extreme discriminating power of antibodies to recognize an almost infinite array of antigenic structures, the application of ELISA to analyte measurement is almost unlimited. ELISAs have been developed in many configurations depending on the particular application of the assay.
In solid-phase ELISA, one of the immunoreactants (antibody or antigen) is immobilized onto a solid support (microtiter plate) by adsorption, through non-covalent interactions. The immobilized antibody is then incubated with test solution containing the analyte of interest. Following a period of incubation and washing, the bound antigen is detected, by the addition of an enzyme-conjugated antibody that binds to the remaining antigenic sites on the antigen.
Although the technique is easy to perform and quite sensitive, there are certain problems to be solved before it becomes widely usable. In the present Memorandum the technical details are given and the advantages and shortcomings of the procedure are discussed. Present applications and future prospects are reviewed.
OTECHNOLOGY IS CHALLENGING SUBJECT TO TEACH AND UNDERSTAND ALSO .....THEIR INTERESTING PART IS TO LEARN ABOUT MICROBIAL GENETICS AND THEIR METHODS OF GENE TRANSFER
We are engaged in manufacturing, supplying and exporting of Laboratory Filtration Products (syringe filters, membrane filters, glass fiber filters and filter papers). These filtration products are precisely manufactured by making use of superior quality raw material and ultra-modern techniques by the experience team. Our offered products are widely used in various laboratories for various filtration works in worldwide. http://www.axivasichem.com/syringe-filters.aspx
Immobilization of proteins on the solid support of nitrocellulose membrane or polyvinylidinefluoride membrane. Then antibodies bind speciffcally that can be analyzed through Autoradiography
Introduction and Description to Western Blotting, Steps involved in Western Blotting- Sample Preparation, Protein Gel Electrophoresis, SDS-PAGE, Protein Transfer, Electrophoretic Protein Transfer, Transfer Sandwich Diagram, Blocking, Antibody Probing and Detection, Applications of Western Blotting.
Western blotting is a laboratory technique used to detect a specific protein in a blood or tissue sample. The method involves using gel electrophoresis to separate the sample's proteins. The separated proteins are transferred out of the gel to the surface of a membrane.
Following is my journal documentation during Master's in Biotechnology completed in 2015. I do understand many changes would've occurred in the curriculum since then, but the basics seldom change. Kindly absorb as per your need.
Cell migration, a key property of live cells, is the process by which cells move from one location to another. There are numerous ways to study cell migrations. Creative Proteomics offers tailored cell migration services and powerful analysis for your research.
https://www.creative-proteomics.com/services/cell-migration-assay.htm
A brief introfuction of label-free protein quantification methodsCreative Proteomics
If you want to know more about our services, please visit https://www.creative-proteomics.com/services/label-free-quantification.htm.
Label-free protein quantification is a mass spectrometry-based method for identifying and quantifying relative changes in two or more biological samples instead of using a stable isotope-containing compound to label proteins.
If you want to know more, please visit https://www.creative-proteomics.com/s...
Stable isotope labeling using amino acids in cell culture (SILAC) is a powerful method based on mass spectrometry that identifies and quantifies relative differential changes in protein abundance. First used in quantitative proteomics in 2002, it provides accurate relative quantification without any chemical derivatization or manipulation.
Mass Spectrometry-Based Proteomics Quantification: iTRAQ Creative Proteomics
For more information, please visit: https://www.creative-proteomics.com/services/itraq-based-proteomics-analysis.htm
iTRAQ (isobaric tag for relative and absolute quantitation), is an isobaric labeling method to determine the amount of proteins from different sources in just one single experiment by mass spectrometry, which was developed by Applied Biosystems Incorporation in 2004.
If you want to know more, please visit https://www.creative-proteomics.com/services/short-chain-fatty-acids-analysis-service.htm. Short chain fatty acids (SCFAs) are defined as fatty acids with two to six carbon atoms. SCFAs have a wide range of metabolic effects. And SCFA profiling has been a major topic in gut bacteria studies.
For more information, you can visit https://www.creative-proteomics.com/services/protein-post-translational-modification-analysis.htm. In this video, we introduce some commonly used methods to detect PPIs and techniques for proteome-scale interactome maps.
Brief Introduction of Protein-Protein Interactions (PPIs)Creative Proteomics
For more information, please visit https://www.creative-proteomics.com/services/protein-protein-interaction-networks.htm. Protein-protein interactions play important roles in various biological processes. PPIs can be classified based on different factors, including composition, affinity, and lifetime.
Peptidomics represents a short version of “peptide proteomics", which means the comprehensive visualization and analysis of small polypeptides, thus covering the mass range between proteomics and metabonomics.
Mass Spectrometry-based Peptidomics for Biomaker DiscoveryCreative Proteomics
Biomarkers are molecules that indicate a physiological state and also the change during a disease process. In human bodies, peptidome biomarkers can be used to forecast disease, diagnose various disorders, guide clinical therapy, and monitor medicine response. The mass spectrometry-based peptidomics for biomarker discovery contains sample preparation, separation, detection and identification, quantitative evaluation, data analysis, as well as biomarkers validation.
Protein phosphorylation, a reversible process, is characterized by adding phosphate donated from ATP and removing phosphate from a phosphorylated protein substrate. For more information, please visit: https://www.creative-proteomics.com/services/phosphorylation.htm
Protein acetylation commonly has two different forms. In humans, almost (80%-90%) proteins become co-translationally acetylated at their Nα-termini of the nascent polypeptide chains. Another type is typically acetylated on lysine residues.
Mass spectrometry (MS) is the suitable method for the analysis of protein modifications because it can provide universal information about protein modifications without a priori knowledge and locating the sites of modification.
If you are interested in our services, please visit: https://www.creative-proteomics.com/services/protein-post-translational-modification-analysis.htm
Brief introduction of post-translational modifications (PTMs)Creative Proteomics
PTMs are chemical alterations to protein structure, typically catalyzed by exceedingly substrate-specific enzymes, which themselves are under strict control by PTMs. They generate a large diversity of gene products because many types of PTMs are covalently attached to amino-acid residues in each protein. For protein post-translational modification analysis at Creative Proteomics, please visit https://www.creative-proteomics.com/services/protein-post-translational-modification-analysis.htm
Glycomics, the study of glycans, is applied to biology and chemistry that focuses on the structure and function of carbohydrates, and on glycoform distributions at the cellular, tissue, organ and organism levels. Mass spectrometry plays an important role in glycomics analysis. If you want to know more, please visit https://www.creative-proteomics.com/services/glycomics-service.htm
Two-dimensional gel electrophoresis (2-DE) is considered a powerful tool for proteomics work. 2-DE separates proteins depending on two differ steps: the first one is called isoelectric focusing (IEF) which separates proteins according to isoelectric points (pI); the second step is SDS-polyacrylamide gel electrophoresis (SDS-PAGE) which separates proteins based on the molecular weights.
Our website: www.creative-proteomics.com
Membrane proteins play important roles in various cellular processes, such as cell adhesion, immune response, metabolism and signal transduction. They are popular targets for proteomics research and the common candidates for drug development. Shotgun proteomics methods are available for the identification of membrane proteins.
The de novo peptide sequencing is a method for peptide sequencing performed without prior knowledge of the amino acid sequence. It uses computational approaches to deduce the sequence of peptide directly from the experimental MS/MS spectra.This method can obtain the peptide sequences without a protein database. It can be used for un-sequenced organisms, antibodies, peptides with posttranslational modifications, and endogenous peptides.
Proteomics studies play an increasing role in the field of biology. The use of mass spectrometry (MS) in combination with a range of separation methods is the main principal methodology for proteomics. The two principal approaches to identifying and characterizing proteins using MS are the “bottom-up”, which analyze peptides by proteolytic digestion, and “top-down”, which analyze intact proteins.
Peptide mass fingerprinting is a technology to identify proteins. It is a high throughput protein identification technique in which the mass of an unknown protein can be determined. PMF is always performed with MALDI-TOF mass spectrometry
Introduction of mass spectrometer - basic types of ion sourceCreative Proteomics
As we know before, the mass spectrometry consists of three main components, the ion source, the mass analyzer, and the detector. In ion source, a sample is ionized. Today, we are going to introduce several types of ion source, which are usually used in a mass spectrometry.
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
Thanks...!
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
This presentation explores a brief idea about the structural and functional attributes of nucleotides, the structure and function of genetic materials along with the impact of UV rays and pH upon them.
2. Western blot was introduced in 1979,
which is a commonly used method for
protein analysis.
It can be used for qualitative and semi-
quantitative protein analysis.
THREE ELEMENTS
• Separation of proteins by size
• Transferring proteins to a solid support
• Marking proteins by primary and secondary antibodies
for visualization
Western Blot
3. Principles
SDS-PAGE allows protein samples to be separated and transferred to a solid
support. The solid support can absorb the protein and keep its biological
activity unchanged.
Only the proteins to be studied can specifically bind to the primary antibody
to form an antigen-antibody complex.
The labeled secondary antibody that binds to the primary antibody forms
an antibody complex that can indicate the location of the primary antibody,
both the location of the protein being studied.
5. Sample preparation
Cells Tissues
Lysate/Extracts
Proteins can be extracted from different samples, such
as tissues or cells.
Tissues are first broken down by the mechanical
invention, such as homogenizer or sonication.
Protease and phosphatase inhibitors are commonly used
to prevent the digestion of the sample at cold
temperatures.
Detect the concentration of proteins: a
spectrophotometer .
6. Gel electrophoresis
-
+
-
+
High Molecular weight
Low Molecular weight
Stacking gel
Separating gel
The most commonly used gel is polyacrylamide gels (PAG) and buffers loaded with sodium dodecyl
sulfate (SDS).
The smaller the known weight of proteins is, the higher percentage of gels should be used.
5 to 2,000 kDa
7. Proteins transfer
Sponge
Filter Papers
Blotting Membrane
Gel
Filter Papers
Sponge
+
Electroblotting, which uses an electric field oriented perpendicular
to the surface of the gel, to pull proteins out of the gel and move
into the membrane.
The membrane is placed between the gel surface and filter.
The transfer sandwich is created as follows: a sponge, filter
papers, the gel, a membrane, filter papers, a sponge.
Proteins are moved from within the gel onto a solid support
membrane made of nitrocellulose (NC) or polyvinylidene
difluoride (PVDF) to make the proteins accessible to antibody
detection.
Wet conditions are usually more reliable as it is less likely dry out
the gel.
8. Blocking
Prevent antibodies from binding to the membrane
nonspecifically.
The most commonly used typical blockers are
BSA and nonfat dry milk.
When the membrane is placed in the dilute
solution of proteins, the proteins attach to all
places in the membrane where the target proteins
have not attached.
The “noise” in the final product of the western blot
can be reduced and result in clearer results.
9. Antibody incubation
Membrane Membrane
Primary antibody
Membrane
Labeled
secondary antibody
The primary antibody binds to target proteins when the primary antibody is incubated with
the membrane.
The choice of a primary antibody depends on the antigen to be detected.
Washing the membrane with the antibody-buffer solution is helpful for minimizing
background and removes unbound antibodies.
After rinsing the membrane, the membrane is exposed to the specific enzyme-conjugated
secondary antibody.
When performing secondary antibody incubation, the labeled secondary antibody can bind
to the primary antibody which has reacted with target proteins.
10. Protein detection and visualization
Colorimetric
detection
Chemiluminescent
detection
Radioactive
detection
Fluorescent
detection
A substrate reacts with the enzyme that is bound to the secondary antibody to
generate colored substance. It enables us to know the densitometry and location of the
targets protein.
And the size approximations are taken by comparing the proteins bands to the marker.
Electrochemiluminescence
(ECL) system
11. Option 03
Western Blot & Electrical
Transfer
Option 04
2D Blue Native for Complex
Analysis
Option 01
2D Electrophoresis
Option 02
SDS-PAGE, IEF and native
PAGE analysis
Protein Gel
and
Imaging
Analysis
Our services
At Creative Proteomics, we can provide an integrated solution for the identification of low abundance proteins in
complex biological samples.
Western blot was introduced in 1979, which is a commonly used method for protein analysis. It can be used for qualitative and semi-quantitative protein analysis. For the accomplishment of the western blotting, there are three elements, separation of proteins by size, transferring proteins to a solid support, and marking proteins by primary and secondary antibodies for visualization.
Western blot is performed using polypropylene gel electrophoresis. In this method, proteins can be detected, the "probe" is an antibody, and the secondary antibody is used for coloration. SDS-PAGE allows protein samples to be separated and transferred to a solid support, such as nitrocellulose (NC) or polyvinylidene difluoride (PVDF) membrane. The solid support can absorb the protein and keep its biological activity unchanged. The transferred solid support membrane is called a blot and is treated with a protein solution to block the hydrophobic binding site on the membrane. The membrane is treated with the antibody (primary antibody) of the target proteins. Only the proteins to be studied can specifically bind to the primary antibody to form an antigen-antibody complex. After the primary antibody is washed and removed, only the position of the target protein binds to the primary antibody. The primary antibody-treated membranes are treated with a labeled secondary antibody after washing. After treatment, the labeled secondary antibody that binds to the primary antibody forms an antibody complex that can indicate the location of the primary antibody, both the location of the protein being studied.
There are six steps involved in western blot, including sample preparation, gel electrophoresis, proteins transfer, blocking, antibody incubation, and proteins detection and visualization.
Proteins can be extracted from different samples, such as tissues or cells. Since tissue samples display a higher degree of structure, the tissues are first broken down by the mechanical invention, such as homogenizer or sonication. Protease and phosphatase inhibitors are commonly used to prevent the digestion of the sample at cold temperatures. After protein extraction, it is important to detect the concentration of proteins, which permits the mass of proteins loaded into each well. And a spectrophotometer is often used for proteins concentration.
Speaking of the gel electrophoresis,the most commonly used gel is polyacrylamide gels and buffers loaded with sodium dodecyl sulfate. Western blot uses two types of agarose gel: stacking gel that is used for concentrate all proteins in one band, and separating gel, that allows to separating proteins according to their molecular weight. Smaller proteins migrate faster in SDS-PAGE (SDS polyacrylamide gel electrophoresis) when a voltage is applied. PAGE can separate proteins ranging from 5 to 2,000 kDa according to the uniform pore size which is controlled by the Different concentration of PAG. When we choose the appropriate percentage of the separating gel, we should consider the size of the target proteins. The smaller the known weight of proteins is, the higher percentage of gels should be used.
After separating proteins by gel electrophoresis, proteins are moved from within the gel onto a solid support membrane made of nitrocellulose (NC) or polyvinylidene difluoride (PVDF) to make the proteins accessible to antibody detection. The main method for transferring proteins is called electroblotting, which uses an electric field oriented perpendicular to the surface of the gel, to pull proteins out of the gel and move into the membrane. It can be done semi-dry or wet conditions, while wet conditions are usually more reliable as it is less likely dry out the gel. As shown in the left figure, the membrane is placed between the gel surface and filter. The transfer sandwich is created as follows: a fiber pad (sponge), filter papers, the gel, a membrane, filter papers, a fiber pad.
Blocking is an important step in the Western Blot to prevent antibodies from binding to the membrane nonspecifically. The most commonly used typical blockers are BSA and nonfat dry milk. When the membrane is placed in the dilute solution of proteins, the proteins attach to all places in the membrane where the target proteins have not attached. In this way, the “noise” in the final product of the western blot can be reduced and result in clearer results.
After blocking, the primary antibody binds to target protein on the membrane when the primary antibody is incubated with the membrane. The choice of a primary antibody depends on the antigen to be detected. Washing the membrane with the antibody-buffer solution is helpful for minimizing background and removes unbound antibodies. After rinsing the membrane, the membrane is exposed to specific enzyme conjugated the secondary antibody. When performing secondary antibody incubation, the label secondary antibody can bind to the primary antibody which has reacted with target proteins. Based on the species of the primary antibody, we can choose the appropriate secondary antibody.
A substrate reacts with the enzyme that is bound to the secondary antibody to generate colored substance to enable to know the densitometry and location of the targets protein. And the size approximations are taken by comparing the proteins bands to the marker. There are several detection systems are available for protein visualization, such as colorimetric detection, chemiluminescent detection, radioactive detection, and fluorescent detection .The electrochemiluminescence (ECL) system is the most common detection methods.
The western blot is commonly used for qualitative detection of proteins and post-translational modifications (e.g. phosphorylation). In addition, it also can be used in medical diagnostics, such as the HIV-test or BSE-test.
At Creative Proteomics, we can provide an integrated solution for the identification of low abundance proteins in complex biological samples. We can provide two dimensional Electrophoresis, SDS-PAGE, IEF and native PAGE analysis, Western Blot and Electrical Transfer service, 2D Blue Native / SDS-PAGE for Complex Analysis
Thanks for watching our video. At creative proteomics, we provide the most reliable serivices. If you have any questions or specific requirements. Please do not hesitate to contact us. We are very glad to cooperate with you.