1. Immunoprecipitation is a technique used to isolate a protein of interest along with any interacting proteins from a mixture. It involves using an antibody against the target protein to form an immune complex, which is then isolated using a secondary antibody or protein A/G.
2. Immunogold labeling localizes proteins in cells and tissues using antibodies tagged with gold particles and visualized with electron microscopy. It was used in one study to quantify serotonin receptor subtypes in post-mortem brain samples from suicide victims versus controls.
3. Western blotting separates proteins by gel electrophoresis then transfers them to a membrane where antibodies are used to detect specific proteins. It allows visualization and quantification of proteins.
Protein targeting or protein sorting is the mechanism by which a cell transports to the appropriate positions in the cell or outside of it. Both in prokaryotes and eukaryotes, newly synthesized proteins must be delivered to a specific sub-cellular location or exported from the cell for correct activity. This phenomenon is called protein targeting. Protein targeting is necessary for proteins that are destined to work outside the cytoplasm.This delivery process is carried out based on information contained in the protein itself. Correct sorting is crucial for the cell; errors can lead to diseases. In 1970, Günter Blobel conducted experiments on the translocation of proteins across membranes. He was awarded the 1999 Nobel Prize for his findings. He discovered that many proteins have a signal sequence, that is, a short amino acid sequence at one end that functions like a postal code for the target organelle.
Protein targeting or protein sorting is the mechanism by which a cell transports to the appropriate positions in the cell or outside of it. Both in prokaryotes and eukaryotes, newly synthesized proteins must be delivered to a specific sub-cellular location or exported from the cell for correct activity. This phenomenon is called protein targeting. Protein targeting is necessary for proteins that are destined to work outside the cytoplasm.This delivery process is carried out based on information contained in the protein itself. Correct sorting is crucial for the cell; errors can lead to diseases. In 1970, Günter Blobel conducted experiments on the translocation of proteins across membranes. He was awarded the 1999 Nobel Prize for his findings. He discovered that many proteins have a signal sequence, that is, a short amino acid sequence at one end that functions like a postal code for the target organelle.
Yeast two hybrid system for Protein Protein Interaction Studiesajithnandanam
Yeast Two Hybrid system uses a reporter gene to detect the interaction of pair of proteins inside the yeast cell nucleus. In the yeast Two Hybrid System, The interaction of target protein to the protein will bring together transcriptional activator, which then switches on the expression of reporter gene.
Yeast two hybrid system for Protein Protein Interaction Studiesajithnandanam
Yeast Two Hybrid system uses a reporter gene to detect the interaction of pair of proteins inside the yeast cell nucleus. In the yeast Two Hybrid System, The interaction of target protein to the protein will bring together transcriptional activator, which then switches on the expression of reporter gene.
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.
Apoptosis is characterized by several biochemical features including cell shrinkage, membrane blebbing, chromosome condensation, nuclear fragmentation, DNA laddering and the eventual engulfment of the cell by phagosomes.
The main purpose of these slides is to convey information to the Professors, Lecturers, and Students. These slides contain authentic information about this topic which is mentioned in that.
Kubernetes & AI - Beauty and the Beast !?! @KCD Istanbul 2024Tobias Schneck
As AI technology is pushing into IT I was wondering myself, as an “infrastructure container kubernetes guy”, how get this fancy AI technology get managed from an infrastructure operational view? Is it possible to apply our lovely cloud native principals as well? What benefit’s both technologies could bring to each other?
Let me take this questions and provide you a short journey through existing deployment models and use cases for AI software. On practical examples, we discuss what cloud/on-premise strategy we may need for applying it to our own infrastructure to get it to work from an enterprise perspective. I want to give an overview about infrastructure requirements and technologies, what could be beneficial or limiting your AI use cases in an enterprise environment. An interactive Demo will give you some insides, what approaches I got already working for real.
Builder.ai Founder Sachin Dev Duggal's Strategic Approach to Create an Innova...Ramesh Iyer
In today's fast-changing business world, Companies that adapt and embrace new ideas often need help to keep up with the competition. However, fostering a culture of innovation takes much work. It takes vision, leadership and willingness to take risks in the right proportion. Sachin Dev Duggal, co-founder of Builder.ai, has perfected the art of this balance, creating a company culture where creativity and growth are nurtured at each stage.
Essentials of Automations: Optimizing FME Workflows with ParametersSafe Software
Are you looking to streamline your workflows and boost your projects’ efficiency? Do you find yourself searching for ways to add flexibility and control over your FME workflows? If so, you’re in the right place.
Join us for an insightful dive into the world of FME parameters, a critical element in optimizing workflow efficiency. This webinar marks the beginning of our three-part “Essentials of Automation” series. This first webinar is designed to equip you with the knowledge and skills to utilize parameters effectively: enhancing the flexibility, maintainability, and user control of your FME projects.
Here’s what you’ll gain:
- Essentials of FME Parameters: Understand the pivotal role of parameters, including Reader/Writer, Transformer, User, and FME Flow categories. Discover how they are the key to unlocking automation and optimization within your workflows.
- Practical Applications in FME Form: Delve into key user parameter types including choice, connections, and file URLs. Allow users to control how a workflow runs, making your workflows more reusable. Learn to import values and deliver the best user experience for your workflows while enhancing accuracy.
- Optimization Strategies in FME Flow: Explore the creation and strategic deployment of parameters in FME Flow, including the use of deployment and geometry parameters, to maximize workflow efficiency.
- Pro Tips for Success: Gain insights on parameterizing connections and leveraging new features like Conditional Visibility for clarity and simplicity.
We’ll wrap up with a glimpse into future webinars, followed by a Q&A session to address your specific questions surrounding this topic.
Don’t miss this opportunity to elevate your FME expertise and drive your projects to new heights of efficiency.
JMeter webinar - integration with InfluxDB and GrafanaRTTS
Watch this recorded webinar about real-time monitoring of application performance. See how to integrate Apache JMeter, the open-source leader in performance testing, with InfluxDB, the open-source time-series database, and Grafana, the open-source analytics and visualization application.
In this webinar, we will review the benefits of leveraging InfluxDB and Grafana when executing load tests and demonstrate how these tools are used to visualize performance metrics.
Length: 30 minutes
Session Overview
-------------------------------------------
During this webinar, we will cover the following topics while demonstrating the integrations of JMeter, InfluxDB and Grafana:
- What out-of-the-box solutions are available for real-time monitoring JMeter tests?
- What are the benefits of integrating InfluxDB and Grafana into the load testing stack?
- Which features are provided by Grafana?
- Demonstration of InfluxDB and Grafana using a practice web application
To view the webinar recording, go to:
https://www.rttsweb.com/jmeter-integration-webinar
UiPath Test Automation using UiPath Test Suite series, part 4DianaGray10
Welcome to UiPath Test Automation using UiPath Test Suite series part 4. In this session, we will cover Test Manager overview along with SAP heatmap.
The UiPath Test Manager overview with SAP heatmap webinar offers a concise yet comprehensive exploration of the role of a Test Manager within SAP environments, coupled with the utilization of heatmaps for effective testing strategies.
Participants will gain insights into the responsibilities, challenges, and best practices associated with test management in SAP projects. Additionally, the webinar delves into the significance of heatmaps as a visual aid for identifying testing priorities, areas of risk, and resource allocation within SAP landscapes. Through this session, attendees can expect to enhance their understanding of test management principles while learning practical approaches to optimize testing processes in SAP environments using heatmap visualization techniques
What will you get from this session?
1. Insights into SAP testing best practices
2. Heatmap utilization for testing
3. Optimization of testing processes
4. Demo
Topics covered:
Execution from the test manager
Orchestrator execution result
Defect reporting
SAP heatmap example with demo
Speaker:
Deepak Rai, Automation Practice Lead, Boundaryless Group and UiPath MVP
Transcript: Selling digital books in 2024: Insights from industry leaders - T...BookNet Canada
The publishing industry has been selling digital audiobooks and ebooks for over a decade and has found its groove. What’s changed? What has stayed the same? Where do we go from here? Join a group of leading sales peers from across the industry for a conversation about the lessons learned since the popularization of digital books, best practices, digital book supply chain management, and more.
Link to video recording: https://bnctechforum.ca/sessions/selling-digital-books-in-2024-insights-from-industry-leaders/
Presented by BookNet Canada on May 28, 2024, with support from the Department of Canadian Heritage.
GraphRAG is All You need? LLM & Knowledge GraphGuy Korland
Guy Korland, CEO and Co-founder of FalkorDB, will review two articles on the integration of language models with knowledge graphs.
1. Unifying Large Language Models and Knowledge Graphs: A Roadmap.
https://arxiv.org/abs/2306.08302
2. Microsoft Research's GraphRAG paper and a review paper on various uses of knowledge graphs:
https://www.microsoft.com/en-us/research/blog/graphrag-unlocking-llm-discovery-on-narrative-private-data/
UiPath Test Automation using UiPath Test Suite series, part 3DianaGray10
Welcome to UiPath Test Automation using UiPath Test Suite series part 3. In this session, we will cover desktop automation along with UI automation.
Topics covered:
UI automation Introduction,
UI automation Sample
Desktop automation flow
Pradeep Chinnala, Senior Consultant Automation Developer @WonderBotz and UiPath MVP
Deepak Rai, Automation Practice Lead, Boundaryless Group and UiPath MVP
DevOps and Testing slides at DASA ConnectKari Kakkonen
My and Rik Marselis slides at 30.5.2024 DASA Connect conference. We discuss about what is testing, then what is agile testing and finally what is Testing in DevOps. Finally we had lovely workshop with the participants trying to find out different ways to think about quality and testing in different parts of the DevOps infinity loop.
Software Delivery At the Speed of AI: Inflectra Invests In AI-Powered QualityInflectra
In this insightful webinar, Inflectra explores how artificial intelligence (AI) is transforming software development and testing. Discover how AI-powered tools are revolutionizing every stage of the software development lifecycle (SDLC), from design and prototyping to testing, deployment, and monitoring.
Learn about:
• The Future of Testing: How AI is shifting testing towards verification, analysis, and higher-level skills, while reducing repetitive tasks.
• Test Automation: How AI-powered test case generation, optimization, and self-healing tests are making testing more efficient and effective.
• Visual Testing: Explore the emerging capabilities of AI in visual testing and how it's set to revolutionize UI verification.
• Inflectra's AI Solutions: See demonstrations of Inflectra's cutting-edge AI tools like the ChatGPT plugin and Azure Open AI platform, designed to streamline your testing process.
Whether you're a developer, tester, or QA professional, this webinar will give you valuable insights into how AI is shaping the future of software delivery.
Epistemic Interaction - tuning interfaces to provide information for AI supportAlan Dix
Paper presented at SYNERGY workshop at AVI 2024, Genoa, Italy. 3rd June 2024
https://alandix.com/academic/papers/synergy2024-epistemic/
As machine learning integrates deeper into human-computer interactions, the concept of epistemic interaction emerges, aiming to refine these interactions to enhance system adaptability. This approach encourages minor, intentional adjustments in user behaviour to enrich the data available for system learning. This paper introduces epistemic interaction within the context of human-system communication, illustrating how deliberate interaction design can improve system understanding and adaptation. Through concrete examples, we demonstrate the potential of epistemic interaction to significantly advance human-computer interaction by leveraging intuitive human communication strategies to inform system design and functionality, offering a novel pathway for enriching user-system engagements.
2. HISTOCHEMICAL METHODS- the application of
physical or chemical methods of analysis to
identify and localize the chemical substances
present in their normal sites in cells/tissues.
CARBOHYDRATES: PAS technique identifies
a number of polysaccharides
and carbohydrate-containing
compounds
LIPIDS: Sudan IV and Sudan
black confer red and
black colors on lipids
DNA: presence is
detected by Feulgen
reaction
3. PROTEIN ISOLATION
Proteins can be separated from other cell components and
from one another on the basis of differences in their
physical and chemical properties.
1. Gel electrophoresis separates proteins on the basis of
their rates of movement in an applied electric field.
SDS polyacrylamide gel electrophoresis can resolve
polypeptide chains differing in molecular weight by
10% or less.
2. Centrifugation separates proteins on the basis of their
rates of sedimentation, which are influenced by their
masses and shapes.
3. Chromatography separates proteins on the basis of
their rates of movement through a column packed with
spherical beads. Proteins differing in mass are
resolved on gel filtration columns; those differing in
charge, on ion exchange columns; and those differing
in ligand-binding properties, on affinity columns.
4. SDS polyacrylamide gel electrophoresis
(left) separates proteins solely on the
basis of their masses.
Two-dimensional gel electrophoresis
(right) can separate proteins of similar
mass.
7. Use of MASS SPECTROMETRY to Tryptic peptides are first
identify proteins and to sequence separated based on mass
peptides. In the first method, within a mass spectrometer.
peptide masses are measured and Each peptide is then further
sequence databases are then fragmented primarily by
searched to find the gene that cleaving its peptide bonds.
encodes a protein whose Repeated applications of
calculated tryptic digest profile determining mass
matches these values. differences yield protein
partial amino acid sequence.
8. ISOELECTRIC FOCUSING. At low pH, the carboxylic acid groups of
proteins tend to be uncharged and their nitrogen-containing basic
groups fully charged giving most proteins a net positive charge. At high
pH, he carboxylic acid groups are negatively charged and the basic
groups tend to be uncharged, giving most proteins a net negative
charge. At its isoelectric pH, a protein has no net charge since the
positive and negative charges balance. Thus when a tube containing a
fixed pH gradient is subjected to a strong electric field in the
appropriate direction, each protein species present migrates until it
forms a sharp band at its isoelectric pH.
9. COLUMN CHROMATOGRAPHY
• The sample, a mixture of different molecules, is
applied to the top of a cylindrical glass or plastic
column filled with a permeable solid matrix, such as
cellulose, immersed in solvent.
• A large amount of solvent
is then pumped slowly
through the column and
collected in separate
tubes as it emerges from
the bottom.
• Because various
components of the
sample travel at different
rates through the column,
they are fractionated into
different tubes.
10. ION-EXCHANGE CHROMATOGRAPHY
(A) the insoluble matrix carries ionic charges that retard the
movement of molecules of opposite charge. The strength of
the association between the dissolved molecules and the ion-
exchange matrix depends on both the ionic strength and the
pH of the solution that is passing down the column, which may
therefore be varied systematically to achieve an effective
separation. (B) In gel-filtration chromatography, the matrix is
inert but porous. (C) Affinity chromatography relies on
antigen-antibody interactions.
11. ASSAYS FOR DETECTING AND QUANTIFYING PROTEINS
1.Staining. All proteins will stain the same color but the
color intensity is proportional to the protein
concentration.
2.Autoradiography. An x-ray film is apposed to the gel
for a certain time and then developed. Radioactive
proteins will appear as dark bands in the film and can
be used as a semi-quantitative technique for
detecting molecules in cells, tissues, or gels.
3.Pulse-chase labeling can determine the intracellular
fate of proteins and other metabolites.
4.Generating amplified signals through the use of
fluorescence, enzymes or chromogenic substrates,
and colored probes (gold). Some probes can detect
and measure rapidly changing intracellular ion
concentrations inside cells.
12. 5. Antibodies are powerful reagents used to detect,
quantify, and isolate proteins. They are used in
affinity chromatography and combined with gel
electrophoresis in Western blotting.
6. Immunoblotting. The isolated proteins are
transferred from the gel to a nitrocellulose
membrane. The membrane is incubated with an
antibody made against proteins that may be
present in the sample.
7. 3-D structures of proteins are obtained by x-ray
crystallography (provides the most detailed
structures but requires protein crystallization),
cryoelectron microscopy (most useful for large
protein complexes, which are difficult to
crystallize), and NMR or nanomagnetic resonance
spectroscopy (only relatively small proteins are
amenable to NMR analysis).
13. Shown are schematic
Combined methods for depictions of gels for the
protein isolation, starting mixture of proteins
detection, and purification. (lane 1) and samples taken after
each of several purification
steps. In the first step, salt
fractionation, proteins that
precipitated with a certain
amount of salt were re-
dissolved; electrophoresis of
this sample (lane 2) shows that
it contains fewer proteins than
the original mixture. The sample
then was subjected in
succession to three types of
column chromatography that
separate proteins by electrical
charge, size, or binding affinity
for a particular small molecule
The final preparation is quite
pure, as can be seen from the
appearance of just one protein
band in lane 5.
14. Compounds that have affinity
toward another molecule can
be tagged with a label and used
to identify that molecule.
(1) Molecule A has a high and
specific affinity toward a
portion of molecule B.
(2) When A and B are mixed, A
binds to the portion of B it
recognizes. (3) Molecule A may
be tagged with a label that can
be visualized with a light or
electron microscope. The label
can be a fluorescent
compound, an enzyme such as
peroxidase, a gold particle, or a
radioactive atom.
(4) If molecule B is present in a
cell or extracellular matrix that
is incubated with labeled
molecule A, molecule B can be
detected.
15. AUTORADIOGRAPHY
Radioisotopes are
taken up selectively
by cells to be studied
Exposure of
photographic film to
their emitted radiation
reveal presence of
such isotopes in the
vicinity of these
target cells
Silver bromide
crystals in emulsion
detect radiation, that
reduce them to visible
black granules.
16. Pulse-chase autoradiography, Pancreatic B cells were fed with 3H-
leucine for 5 minutes (the pulse) followed by excess unlabeled leucine
(the chase). The amino acid is largely incorporated into insulin, which
is destined for secretion. After a 10-minute chase the labeled protein
has moved from the rough ER to the Golgi stacks (A), where its
position is revealed by the black silver grains in the photographic
emulsion. After a further 45-minute chase the labeled protein is found
in electron-dense secretory granules (B). The small round silver grains
seen here are produced by using a special photographic developer.
Experiments similar to this were important in establishing the
intracellular pathway taken by newly synthesized secretory proteins.
17. Useful in:
Mapping anatomical location of labelled ligands to
visualize and quantify receptors in tissue
Studying sequence and intensity of events occurring
in tissue components
Measuring DNA production (e.g., 3H-thymidine)
Advantages: protocol is simple & easy to follow
Disadvantages:
Everything binds to everything (misinterpret results)
There are no biochemical or physiological criteria to
assess the binding specificity (i.e., to determine
whether the binding site really corresponds to an
actual receptor)
The presence of a high-affinity labelled receptor
does not necessarily imply that the receptor has
physiological significance
Ligands are not always very specific
18. Methods of introducing a membrane-impermeant substance into a cell
(A) The substance is injected through a micropipette, either by applying pressure or, if the
substance is electrically charged, by applying a voltage that drives the substance into the cell
as an ionic current (a technique called iontophoresis). (B) The cell membrane is made
transiently permeable to the substance by disrupting the membrane structure with a brief but
intense electric shock (2000 V/cm for 200 μsec, for example). (C) Membrane-enclosed vesicles
are loaded with the desired substance and then induced to fuse with the target cells. (D) Gold
particles coated with DNA are used to introduce a novel gene into the nucleus.
19. These techniques “stain” various enzymes within
cells and tissues by making use of the enzyme
activity itself.
The enzyme is made to react with a specific
substrate. The product of this reaction may itself be
visible in the microscope and thus demonstrate the
presence of the enzyme at a specific location,
or the reaction product is
subsequently reacted to form
a visible secondary
reaction product.
Examples:
Acid Phosphatase –
Gomori-Takamatsu method
Peroxidase – DAB method
20. Antigen-antibody
reactions are high-
affinity interactions
It localizes in tissues
the following:
a.antigen-antibody
reactions
b.segments of NA
(hybridization)
c. specific carbohy-
drate moieties
(lectin-binding)
d. macromolecules
(e.g. phalloidin
interacts with actin in microfilaments).
21. 1.Direct method - marker conjugated directly to the
antibody that binds to the molecule we are
interested in.
2.Indirect method - marker bound to antibody that will
bind to the antibody that binds to the molecule we
are interested in (i.e. GAM - IgG).
22. Direct method of immunocytochemistry.
(1) Immunoglobulin molecule (Ig). (2) Production of a
polyclonal antibody. Protein x from a rat is injected into
a rabbit. Several rabbit Igs are produced against protein
x. (3) Labeling the antibody. The rabbit Igs are tagged
with a label. (4) Immunocytochemical reaction. The
rabbit Igs recognize and bind to different parts of
protein x.
23. . Indirect method of immunocytochemistry.
(1) Production of primary polyclonal antibody. Protein x from a rat is
injected into a rabbit. Several rabbit immunoglobulins (Ig) are produced
against protein x. (2) Production of secondary antibody. Ig from a
nonimmune rabbit is injected into a goat. Goat Igs against rabbit Ig are
produced. The goat Igs are then isolated and tagged with a label. (3) First
step of immunocytochemical reaction. The rabbit Igs recognize and bind
to different parts of protein x. This detection method is very sensitive.
Commonly used marker molecules include fluorescent dyes (for
fluorescence microscopy), the enzyme horseradish peroxidase (for either
light microscopy or EM), colloidal gold spheres (for EM), and the enzymes
alkaline phosphatase or peroxidase (for biochemical detection).
24.
25. Photomicrograph of
a section of small
intestine in which an
antibody against the
enzyme lysozyme
was applied to
demonstrate
lysosomes in
macrophages and
Paneth cells. The
brown color results
from the reaction
done to show
peroxidase, which
was linked to the
secondary antibody.
26. Medical applications The
technique of
coupling a
tumor cell
with the
antigen-
antibody
complex has
allowed the
production
of
monoclonal
antibodies
capable of
treating
specific
disorders.
http://highered.mcgraw-hill.com/olc/dl/120110/micro43.swf
27. Hybridoma
cells are
widely used to
produce
unlimited
quantities of
uniform
monoclonal
antibodies
which are also
used to detect
and purify
proteins.
28. The Enzyme-Linked Immunosorbent Assay (ELISA) is
a technique used to detect antibodies or infectious
agents in a sample.
For an antibody ELISA, antigens are stuck onto a plastic surface, a sample is
added and any antibodies for the disease tested for will bind to the antigens.
Next a second antibody with a marker is added and a positive reaction is
detected by the marker changing color when an appropriate substrate is
added. If there are no antibodies in the sample, the second antibody will not
be able to stick and there will be no color change. For an antigen ELISA,
antibodies are bound to a plastic surface, a sample is added and if antigens
from the virus tested for are present, they will stick to the antibodies. This test
then proceeds in the same way as the antibody ELISA.
29. IMMUNOPRECIPITATION
Live specimen is incubated in radioactive amino acids
Total proteins are extracted and
incubated with specific antibody
Antibody will bind to its target protein
and form an immune complex,
Antigen-Antibody complex is incubated with protein A
(bacterial protein that binds tightly to IgG-type antibodies)
The bound antibody and target protein are
run on a protein gel, and the radioactive
band of target protein is visualized
30. Applications of Immunoprecipitation: Determination of the molecular weight
and quantity of immunoprecipitated protein; assess for protein-protein
interactions, done by immunoprecipitation for one protein, and then blotting
for another protein; quantification of rate of synthesis of a protein in cells by
determining the quantity of radio-labeled protein made during a specific
amount of time; concentrate proteins that are otherwise difficult to detect.
31. At the top is a thin
section of a yeast
mitotic spindle showing
spindle microtubules
that cross the nucleus,
connecting at each end
to spindle pole bodies
embedded in the NE.
Below are components
of a single spindle pole
body. Antibodies
SIGNAL against 4 different
proteins of the spindle
AMPLIFICATION: pole body are used,
IMMUNOGOLD together with colloidal
gold particles (black
dots), to reveal where
within the complex
structure each protein
is located.
32. Immunogold Labelling of Serotonin
In this study,
Receptors in Suicide Victims immunogold
Control Suicide labelling was used
to quantify the
density of 5-HT2A
and 5-HT2C
subtypes of
serotonin
5-HT2A
receptors in the
PFC of suicide
victims and
controls. It was
found that in
suicide victims,
there is a
significant
increase in 5-HT2A,
5-HT2C but not 5-HT2C
receptors on
pyramidal cells of
cortical layer III.
33. Total proteins of the sample are extracted
and separated on a protein gel
Proteins are blotted on a membrane
incubated with a specific antibody.
The bound antibody is then visualized with a 2nd
antibody directed against the 1st antibody
Complex is modified for easy detection (e.g.
radioactive labeling, conjugating with enzymes that
produce intensely colored and insoluble reaction
products with substrates)
After incubation, a colored precipitate will form on
the membrane, corresponding to the position and
quantity of the target protein in the original sample
34.
35. Lane 1 is a protein size marker ladder which shows
different known sizes of proteins, Lane 3 is a cancer
sample & lane 5 is a normal sample. Lanes 3 & 5 are the
same size as the 2nd spot in the size ladder from lane 1.
36.
37. Green Fluorescent Protein (GFP)
GFP is an especially versatile probe that can be attached
to other proteins by genetic manipulation.
Variants have been generated with altered absorption
and emission spectra in the blue-green-yellow range. A
family of related fluorescent proteins has been
discovered in corals, extending the range into the red
region of the spectrum.
Virtually any protein of interest can be
genetically engineered as a GFP-fusion
protein, and then imaged in living cells by
fluorescence microscopy.
Peptide location signal can also be added to
GFP to direct it to a particular cellular
compartment, such as the ER or a mitochondrion,
lighting up these organelles so they can be observed in
the living state. GFP is also used as a reporter molecule
to monitor gene expression.
38. (A) The upper surface of
the leaves of Arabidopsis
plants are covered with
huge branched single-cell
hairs that rise up from the
surface of the epidermis.
These hairs, or trichomes,
can be imaged in the SEM.
(B) If an Arabidopsis plant
is transformed with a DNA
sequence coding for talin (an actin-binding protein), fused
to a DNA sequence coding for GFP, the fluorescent talin
protein produced binds to actin filaments in all the living
cells of the transgenic plant.
Confocal microscopy can reveal the dynamics of the
entire actin cytoskeleton of the trichome (green). The red
fluorescence arises from chlorophyl in cells within the leaf
below the epidermis.
39. Lectin Histochemistry
• Lectins are proteins derived from plant seeds
• They are membrane-bound carbohydrate-
binding proteins that bind to specific
sequences of
cell-surface
carbohydrate
residues on both
glycolipids and
glycoproteins in
the process of
cell-cell adhesion
40. Fluorescence microscopy of a human skin tissue section (paraffin
fixation) with fungal infection. The target carbohydrate subunit
chitotriose [(GlcNAc)3] of the pathogenic fungi are specifically
bound to lectin from Phytolacca americana-Atto 488 conjugate
(green). The nuclei are counterstained with DAPI (blue).
41. ION-SENSITIVE INDICATORS
Rapidly changing intracellular ion
concentrations can be measured
with light-emitting indicators
Their light emission reflects the
local concentration of the ion are
used to record rapid and transient
changes in cytosolic ion
concentration.
Some of these indicators are
luminescent, while others are
fluorescent. Aequorin is a
luminescent protein isolated from a
marine jellyfish; it emits light in the
presence of Ca2+ and responds to
changes in Ca2+ concentration in
the range of 0.5–10 μM.
42. Fluorescent Indicator Dyes
They can be introduced to measure the
concentrations of specific ions in individual cells or
in different parts of a cell. Visualizing intracellular Ca2+
concentrations by using a
fluorescent indicator.
The intracellular Ca2+
concentration in a single
Purkinje cell (from the brain of a
guinea pig) was taken with a low-
light camera and the Ca2+-
sensitive fluorescent indicator
fura-2. The concentration of free
Ca2+ is represented by different
colors, red being the highest and
blue the lowest. The highest Ca2+
levels are present in the
thousands of dendritic branches.
43. Caged Precursor
The dynamic behavior of many molecules can be followed in
a living cell by constructing an inactive “caged” precursor,
which can be introduced into a cell and then activated in a
selected region of the cell by a light-stimulated reaction.
Caged molecules. A light-sensitive caged derivative of a molecule
(designated X) can be converted by a flash of UV light to its free, active
form. Small molecules such as ATP can be caged in this way. Even ions
like Ca2+ can be indirectly caged; in this case a Ca2+-binding chelator is
used, which is inactivated by photolysis, thus releasing its Ca2+.
44. Determining microtubule flux in the mitotic
spindle with caged fluorescein linked to tubulin
(A) A metaphase spindle formed in vitro from an
extract of Xenopus eggs has incorporated three
fluorescent markers: rhodamine-labeled tubulin
(red) to mark all the microtubules, a blue DNA-
binding dye that labels the chromosomes, and
caged-fluorescein-labeled tubulin, which is also
incorporated into all the microtubules but is
invisible because it is nonfluorescent until
activated by ultraviolet light. (B) A beam of UV
light is used to uncage the caged-fluorescein-
labeled tubulin locally, mainly just to the left
side of the metaphase plate. Over the next few
minutes (after 1.5 minutes in C, after 2.5
minutes in D), the uncaged fluorescein-tubulin
signal is seen to move toward the left spindle
pole, indicating that tubulin is continuously
moving poleward even though the spindle
(visualized by the red rhodamine-labeled tubulin
fluorescence) remains largely unchanged.
45. X-RAY DIFFRACTION X-ray crystallography provides
diffraction data from which the 3D
structure of a protein or nucleic acid
can be determined.
(a) Basic components of an x-ray
crystallographic determination.
When a narrow beam of x-rays
strikes a crystal, part of it passes
straight through and the rest is
scattered (diffracted) in various
directions. The intensity of the
diffracted waves is recorded on an x-
ray film or with a solid-state
electronic detector.
(b) X-ray diffraction pattern for a
topoisomerase crystal collected on a
solid-state detector. From complex
analyses of patterns like this one, the
location of every atom in a protein
can be determined
An egg of the medaka fish has been injected with aequorin, at the site of sperm entry reveal a wave of release of free Ca2+ into the cytosol from internal stores just beneath the plasma membrane.