Proteomics Techniques and Applications

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Questions and Answers

What role does a housekeeping protein play in protein quantification?

A housekeeping protein serves as a loading control to normalize the signal of the protein of interest across samples.

Describe the basic process involved in the dot blot method.

The dot blot method involves depositing the sample directly onto a membrane, followed by immunodetection without protein separation via gel electrophoresis.

What is the significance of the sandwich ELISA technique?

The sandwich ELISA technique is significant for detecting a target protein by using a capture antibody and a detection antibody, enabling both qualitative and quantitative analysis.

How do protein microarrays enhance protein detection?

Protein microarrays enhance detection by immobilizing a range of specific antibodies on a slide, allowing simultaneous analysis of multiple protein targets. Signup and view all the answers

What is the primary advantage of using immunostaining for localized detection of proteins?

Immunostaining allows for the qualitative assessment of both the presence and spatial distribution of a target protein within a sample. Signup and view all the answers

Explain the difference between localized and bulk detection methods of protein.

Localized detection methods, such as immunostaining, reveal the spatial distribution of proteins, while bulk detection methods provide overall expression levels without spatial context. Signup and view all the answers

What is the first step in the dot blot method after sample harvest?

The first step after sample harvest is sample denaturation, which prepares the proteins for binding to the membrane. Signup and view all the answers

Why is it important to use both primary and secondary antibodies in immunodetection techniques?

Using both primary and secondary antibodies amplifies the signal, enabling more sensitive detection of the protein of interest. Signup and view all the answers

What is the primary purpose of the forward scatter channel (FSC) in flow cytometry?

The FSC collects data on cell size by gathering light at 0°. Signup and view all the answers

How does the side scatter channel (SSC) contribute to flow cytometry analysis?

The SSC collects data on cell granularity by capturing light at 90°. Signup and view all the answers

What role do fluorescent antibodies play in flow cytometry?

Fluorescent antibodies allow for the excitation of specific cell markers, emitting light that can be measured. Signup and view all the answers

Describe the main difference between surface markers and intracellular markers in flow cytometry.

Surface markers are fully accessible and compatible with live labeling, while intracellular markers require cell permeabilization and are not compatible with live labeling. Signup and view all the answers

What does the histogram modality represent in flow cytometry data analysis?

The histogram modality shows a mono-parametric analysis where signal intensity is on the x-axis and the number of cells is on the y-axis. Signup and view all the answers

What information does a dot plot provide in flow cytometry?

A dot plot represents a multi-parametric analysis, with each dot corresponding to a droplet analyzed and axes showing fluorescence intensity. Signup and view all the answers

How do fluorochromes function in the context of antibody-based labeling in flow cytometry?

Fluorochromes are attached to antibodies and emit light when excited by specific wavelengths, enabling detection of various cell markers. Signup and view all the answers

Why is it important to choose between direct and indirect labeling methods in flow cytometry?

The choice affects the accessibility of target antigens and the compatibility with live cell labeling techniques. Signup and view all the answers

What is the purpose of using the nuclear-ID dual dye method?

To discriminate between viable and non-viable cells using two different fluorescent dyes. Signup and view all the answers

How do live cells appear in the esterase activity and membrane integrity assay?

Live cells exhibit strong green fluorescence due to esterase enzyme activity that modifies the substrate. Signup and view all the answers

What role does ATP play in cell viability assays?

ATP is a marker of viable cells and can be measured to estimate the number of live cells present. Signup and view all the answers

What happens during the ATP assay after cell lysis?

The total ATP amount is measured using luciferin and luciferase, producing light proportional to the ATP concentration. Signup and view all the answers

Describe the MTT assay and its significance.

The MTT assay measures metabolically active cells by adding MTT dye, which enters the cells and is reduced in the mitochondria. Signup and view all the answers

In the nuclear-ID dual dye method, what does the presence of green fluorescence indicate?

It indicates the presence of dead cells, as the green dye labels only non-viable cells. Signup and view all the answers

What is the consequence of using the ATP assay on the sample?

The sample cannot be reused after the ATP assay is performed. Signup and view all the answers

Why is the MTT dye important for assessing cell viability?

The MTT dye is light yellow and permeable, allowing it to indicate metabolic activity within living cells. Signup and view all the answers

What role does RNAse play in the cDNA synthesis process?

RNAse digests the RNA part, allowing the remaining DNA to serve as a template for synthesizing complementary DNA (cDNA). Signup and view all the answers

List the essential components required for the PCR amplification of cDNA.

The essential components are primers, taqPolymerase, dNTPs, and a buffer mix. Signup and view all the answers

Describe the three main steps involved in one PCR amplification cycle.

The three steps are denaturation (>90°C), annealing (≈50°C), and elongation (≈72°C). Signup and view all the answers

What is the significance of the Ct value in quantitative RT-PCR?

The Ct value indicates the cycle at which the fluorescence threshold is reached; a lower Ct suggests a higher initial copy number of the target gene. Signup and view all the answers

How does the SYBR Green method facilitate quantitative RT-PCR?

SYBR Green binds to double-stranded DNA and emits a fluorescent signal, allowing real-time monitoring of DNA amplification. Signup and view all the answers

In traditional RT-PCR, why is a housekeeping control protein like actin used?

Housekeeping control proteins are used to provide a baseline for semi-quantitative analysis of gene expression. Signup and view all the answers

What happens to the amplification reaction when free primers and dNTPs are depleted?

The reaction reaches a plateau where no further amplification occurs due to the lack of necessary components. Signup and view all the answers

Explain how the annealing step in PCR differs from the denaturation step.

The denaturation step separates DNA strands at high temperatures, while the annealing step involves primers binding to the single-stranded DNA at lower temperatures. Signup and view all the answers

What are the two main applications of ISH and what do they target?

ISH applies to RNA (RISH) and DNA (DISH), targeting gene expression patterns and specific genetic sequences respectively. Signup and view all the answers

How is the probe synthesized for ISH and what is its purpose?

The probe is synthesized from ribonucleotides starting from a DNA template, and it serves to detect and hybridize with the target nucleotide sequence. Signup and view all the answers

What is FISH, and how does it differ from standard ISH?

FISH is ISH with DNA probes that are labeled with fluorochromes, allowing for the visualization of specific sequences in the genome. Signup and view all the answers

Why is a negative control necessary in RNA ISH experiments?

A negative control is needed to detect false positives by using a probe that should not bind to the target mRNA, ensuring specificity. Signup and view all the answers

What limitations are associated with using ISH for gene expression analysis?

ISH is not ideal for analyzing large numbers of genes simultaneously due to the need for numerous probes, and low abundance RNA can also be challenging. Signup and view all the answers

Describe the methodology behind RNAscope and its purpose.

RNAscope uses a pair of probes for signal amplification to achieve single RNA molecule marking in tissue or culture samples. Signup and view all the answers

What role do tags play in the probe detection process during ISH?

Tags, which can be radioactive, fluorescent, or biochemical, allow for the recognition of the probe by antibodies or other detection methods. Signup and view all the answers

How can ISH be used to assess treatment effects in a disease scenario?

ISH can reveal changes in gene expression patterns after treatment, indicating therapeutic effects on specific tissues or cell types. Signup and view all the answers

What role do integrins play in cell adhesion to the extracellular matrix?

Integrins bind to extracellular matrix proteins and activate intracellular signaling events, linking the cell's environment to its interior. Signup and view all the answers

How do actin filaments relate to integrins in the process of cell adhesion?

Actin filaments bind to integrins on the intracellular side, serving as anchoring points that mediate adhesion. Signup and view all the answers

List two intermediary proteins involved in cell adhesion and their function.

Talin and vinculin are intermediary proteins that bridge the gap between integrins and the actin cytoskeleton. Signup and view all the answers

What types of ECM proteins can integrins recognize?

Integrins can recognize extracellular matrix proteins such as collagens, laminins, and vitronectin. Signup and view all the answers

Why is it important for integrins to maintain a temporary link to the ECM?

Maintaining a temporary link allows cells to create the necessary force for movement while also being able to detach when needed. Signup and view all the answers

What happens intracellularly when integrins bind to the ECM?

Integrins transmit information of their binding to activate other components within the cell, influencing cytoskeletal adaptation. Signup and view all the answers

Identify one type of cell adhesion mechanism apart from integrins.

E-cadherin is one type of cell adhesion mechanism that facilitates cell-cell adhesion. Signup and view all the answers

How does the nature of a seeding surface affect cell behavior?

The type of surface a cell rests on influences its adhesion properties and can alter its behavior and response. Signup and view all the answers

Study Notes

Cell Signaling Principles

Cell phenotype is defined by gene expression into mRNA and protein, creating unique profiles during differentiation
Development involves growth and patterning, coordinated by cell signaling between different cell types
Cell proliferation, specialization, interaction, and movement are key aspects of coordinated growth

Generating Cell Diversity

Model 1: Cell lineage model - Cell acquire diverse fates early on, relying on intrinsic factors like mRNA and cytokines. Cell division results in content gradients, creating distinct cell identities throughout development.
Model 2: Diversity from specific signals from other cells/surrounding - Surrounding cells and factors influence cell behavior. Individual stimulus can lead to cell differentiation.
Flag model - Cells have inherent diversity potential (e.g., blue, white, or red). Varying signalling concentrations trigger specific gene expression patterns leading to different cell identities. Morphogens are one type of signaling molecules influencing cell differentiation directly.
Direct morphogen gradient - Localized production of an inducer forms a gradient, influencing developing cells.
Indirect morphogen gradient - Localized inhibitor diffusing away from a source creates a gradient, regulating the inducer activity.

Cell Signaling Mechanisms

Receptor-mediated signaling - extracellular molecules interact with receptor proteins, triggering intracellular signaling cascades affecting metabolism, gene expression, and/or cell movement. Examples include MAPK pathways. Binding can cause activation or signal silencing.
Direct contact - Cell-cell signaling via direct contact between cells influencing neighboring areas. This is very important for generating cell types in development.
Diffusion - Transmission of molecules/signals
Gap junctions - Direct communication channels between cells.

Different Ways of Transmitting Signals

Diffusion of molecules or electric signals via pores and receptors
Direct contact between 2 cells
Gap junctions

Signaling Pathways

Wnt pathway (β-catenin) - Wnt signaling molecules activate the pathway, releasing β-catenin, which moves to the nucleus and modifies gene expression. This controls body axis in normal development and is involved in midbrain and hematopoiesis development.
TGF-β pathway (BMPs/GDF) - Receptors bind and trigger a phosphorylation cascade involving Smads (e.g., Smad2/3), leading to target gene transcription. This pathway is important for body asymmetry, skeleton development, and also neuronal differentiation.
Hedgehog pathway - Signals regulate the hedgehog protein, initiating a cascade involving patched, smoothened and Gli/Ci proteins. Critical for development including neural tube development, axis formation and left-right symmetry.

Cell Proliferation Analysis

Somatic cell division follows G0, G1, S, G2, and M phases, requiring cyclin-CDK complexes for regulation.
DNA strands duplicate during S phase.
Telomeres shorten with each division, posing a limit for self-replication. Telomerase can re-synthesize telomeres.
Cells that stop dividing are in a state of quiescence. Physiological factors like metabolic stimuli, confluence and mechanical stimuli play a role.

Cell Cycle Analysis

Propidium iodide (PI) DNA staining is used to detect DNA content per cell which helps analyse the cell cycle profiles.
Flow cytometry allows cell cycle phase analysis by determining DNA content.
The increase in fluorescence intensity of PI reflects the proportion of cells in different cell cycle phases.
Detection of nucleotide incorporation into DNA during the S phase can identify cells actively dividing. Methods such as BrdU, IdU, and CldU, incorporated are non-radioactive alternatives to the more complicated radioactive thymidine assays.
Analyzing cell cycle progression provides critical information about cellular proliferation dynamics under both normal and pathological conditions.

Proliferation Marker Analysis

PCNA (Proliferating Cell Nuclear Antigen) marker is used for proliferation stage.
Ki-67 marks cycling cells in G1, S, G2, and M phases, while pHH3 (phosphorylated histone H3) identifies mitotic cells (M phase).
Dye dilution assays (e.g., CFSE) can track cell division by dilution of the fluorescent dye, providing information about the proliferation rate or cell division frequency in a cell population.

Protein Marker Detection Techniques

Proteomics - Protein identification uses mass spectrometry following extraction, digestion, and separation of proteins and peptides. Using libraries of known proteins, unknown proteins can then be identified and quantified.
Immunological methods
Western blot - Used to detect and quantify a specific protein target among a mixture.
Dot-blot - No gel electrophoresis, and used for detection of proteins (similar to Western blot but without separation in a gel).
ELISA - Used for qualitative and quantitative readout of proteins in a sample.
Immunostaining - used for spatially localizing proteins in tissue specimens.

Single-cell techniques for protein detection

Flow Cytometry - Measures physical and fluorescent characteristics (cell size, granularity) of individual cells flowing through a laser beam.
Antibody-based labeling in flow cytometry
Allows identifying and quantifying particular cells based on expressing specific markers
Compatible/incompatible with live cell labelling, based on how the antibody needs to get into the cells for the measurement (fully accessible/requires permeability)

How to analyze flow cytometry data

Histograms- Analysing one parameter at a time.
Dot plots- Multi-parameter analysis, where multiple markers/parameters can be analysed.

Cell Sorting with FACS

Fluorescence-Activated Cell Sorting (FACS) - Separates cells based on physical and fluorescent characteristics (size, granularity, fluorescence intensity).

Cell Migration Analysis

Cell migration involves complex interactions with the extracellular matrix and neighboring cells, regulated by signaling and cytoskeletal changes. Several phases are typically involved in cell migration, including initial attachment, cell spreading and stable adhesion.
Factors influencing migration include chemotaxis, cell-cell contact (contact inhibition/stimulation), and changes in cell density.
Assays for measuring cell migration include scratch assays, 2D migration track monitoring, micropillar migration assays and membrane migration essays. Measuring forces in cell migration will help clarify the mechanisms behind it.

Cell Health Assays

Viability assays - Measure the proportion of live cells (including Trypan Blue exclusion, nuclear staining, or esterase activity assays.).
Apoptosis assays - Identify apoptotic cells (including phosphatidylserine, TUNEL, or caspase assays.). Useful in monitoring cells during treatment for understanding and evaluating how efficient the treatments were at driving cell death.

Gene Expression Analysis, RNA Markers

Northern blot - Detects specific RNA molecules in a sample (semi-quantitative).
Quantitative RT-PCR (qRT-PCR) - Measures the amount of RNA for specific genes in a sample (quantitative).
RNA sequencing (RNA-Seq) - A method by which to identify a larger variety of transcripts, and is used as a high-throughput method for analyzing the entire transcriptome.
Microarrays/Nanostring Technology - used to determine the expression levels of a large number of transcripts at one time.

Cell Engineering:

Genetic modifications
Inducible expression - This method is used to turn on or turn off a gene's expression based on the presence or absence of a chemical inducer. This is done via inducible promoters or by placing the promoter under control of an external signal/compound.
Repressing gene expression ("knock down") - siRNAs can target specific mRNA for degradation, reducing its expression. Other methods include using vectors to knock out genes altogether.
Triggering mutations - Site-directed mutagenesis (changing an existing amino acid to a codon that codes for a different amino acid.) and CRISPR-Cas (target-specific mutations).

Transgenesis

Pronuclear microinjection - Foreign genes are introduced into a fertilized embryo to generate transgenic animals.
Blastocyst injection- Genetically modified stem cells are added to a blastocyst to create transgenic offspring.
Calcium phosphate transfection - Used to introduce DNA into cells using a precipitate.
Electroporation - Uses electric pulses to introduce DNA into cells.
Viral transduction - Viruses carrying foreign genes are used to infect and transfer the DNA into the cells.

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Description

This quiz explores various techniques in proteomics, including the dot blot method, sandwich ELISA, and flow cytometry. Understand the role of housekeeping proteins, the importance of antibodies in immunodetection, and differences in detection methods. Test your knowledge on how these techniques enhance protein detection and analysis.