Proteomics Techniques and Applications

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

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

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.

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

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

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

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

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

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

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

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

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

<p>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.</p> Signup and view all the answers

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

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

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

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

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

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

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

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

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

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

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

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

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

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

<p>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.</p> Signup and view all the answers

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

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

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

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

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

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

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

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

<p>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.</p> Signup and view all the answers

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

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

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

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

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

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

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

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

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

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

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

A method used to analyze the physical and chemical characteristics of individual cells within a heterogeneous population.

A channel in a flow cytometer that measures the size of cells based on the amount of light scattered forward by the cell.

A channel in a flow cytometer that measures the granularity or internal complexity of a cell based on the amount of light scattered sideways by the cell.

A channel in a flow cytometer that measures the fluorescence intensity of a cell after it has been stained with a fluorescent antibody.

A method of labeling cells with fluorescent antibodies to identify specific cell populations or markers.

A type of cell marker that is located on the outer surface of a cell and can be directly accessed by antibodies.

A type of cell marker that is located inside a cell and requires cell permeabilization to be accessed by antibodies.

A mode of data analysis in flow cytometry where a single parameter (e.g., fluorescence intensity) is analyzed at a time to identify subpopulations of cells.

A method used to quantify proteins of interest by comparing their signal to a reference protein (housekeeping protein), ensuring consistent results across samples.

A technique that directly transfers proteins onto a membrane without gel electrophoresis, allowing for the detection of specific proteins within a sample.

A protein that is consistently expressed in all cells of an organism, serving as a reference for normalizing protein levels in experiments.

A technique that utilizes antibodies and enzyme-linked substrates to quantify the presence of specific proteins in a sample.

A type of ELISA that employs a capture antibody and a detection antibody to sandwich the target protein, allowing for its quantitative detection.

A high-throughput technique that allows for the simultaneous detection of multiple proteins in a sample by immobilizing specific antibodies onto a slide.

A technique used to determine the location of a specific protein within a sample, providing information on its spatial distribution.

The use of antibodies to detect the presence of a specific protein within a sample, providing a qualitative measure of its expression.

A method using two fluorescent dyes to differentiate between live and dead cells. A blue dye stains all cells, while a green dye only enters dead cells with compromised membranes.

This method relies on the activity of intracellular esterase, an enzyme present in live cells, and the integrity of the cell membrane.

A method for determining cell viability based on the presence of ATP, a molecule essential for cellular function.

One of the first methods for assessing cell viability by measuring the activity of metabolically active cells in culture.

A green fluorescent dye used in the Esterase Assay.

A red fluorescent dye used in the Esterase Assay that enters only cells with damaged membranes, staining their DNA.

An enzyme that uses ATP to produce light from luciferin.

A molecule used in the ATP Assay; it interacts with luciferase and ATP to produce light.

A technique that uses an enzyme called reverse transcriptase to convert RNA into DNA (cDNA). This cDNA can then be amplified using PCR.

A technique that uses a chain reaction to amplify a specific DNA sequence, creating many copies. It involves three steps: denaturation, annealing, and elongation, repeated for multiple cycles.

The process of separating the two strands of a DNA double helix by heating it to a high temperature.

The process where primers, short segments of DNA, bind to complementary sequences on the single-stranded DNA template.

The process where DNA polymerase extends the primers, using dNTPs (building blocks of DNA), to synthesize new DNA strands.

A special enzyme that can withstand high temperatures and is used in PCR to amplify DNA sequences.

A technique that measures the amount of a specific DNA sequence in a sample using fluorescent probes. The amount of fluorescence emitted is directly proportional to the amount of DNA present.

A dye that binds to double-stranded DNA and fluoresces under UV light, used in qPCR to measure the amount of DNA amplified.

A technique that uses labeled probes to detect specific DNA or RNA sequences within a sample. This allows for the visualization of gene expression and localization.

ISH that uses labeled DNA probes to detect specific DNA sequences. It's commonly used for genetic diagnostics and research.

ISH that uses labeled RNA probes to detect specific RNA sequences. It allows for the study of gene expression patterns within a sample.

A type of DISH where the DNA probe is labeled with fluorescent molecules, allowing the target sequences to be visualized under a fluorescent microscope.

A specialized RISH technique that uses probe pairs and signal amplification to achieve single RNA molecule detection in a sample.

A probe that binds to the target sequence only if both probes are bound, ensuring accurate detection of the target.

A probe designed to bind to a non-complementary sequence, used as a control in ISH experiments to detect false positive signals. It should not produce any signal if the experiment is working correctly.

The process of adding a tag, such as an antibody or fluorescent molecule, to a probe to make it detectable. This allows for the visualization of the target sequence once the probe has hybridized with the sample.

Integrins are transmembrane proteins that bind to extracellular matrix (ECM) proteins, like collagen and laminin. They act as receptors, sensing and attaching to the ECM, and transmitting signals inside the cell.

Integrins play a crucial role in cell adhesion, helping cells attach to their surroundings. This adhesion is essential for cell migration, tissue formation, and wound healing.

Integrins are linked to actin filaments inside the cell. Actin is a protein that forms filaments, providing structural support and allowing movement within the cell.

Talin and vinculin are proteins that act as 'bridges,' connecting integrins with actin filaments. They help transmit signals from the ECM to the cell.

When integrins bind to ECM, they activate internal signaling pathways, recruiting proteins like talin and vinculin. This process modifies the cell's cytoskeleton, allowing it to adapt its shape and movement to the surrounding environment.

Cell adhesion can occur through different types of junctions, each playing a specific role in cell-cell communication and tissue organization. These junctions include E-cadherin, Occludin/Claudin, and Gap junctions.

The material a cell rests on, or its 'substrate,' can significantly influence its behavior, including its growth, differentiation, and overall functions.

Integrins need to maintain their grip on the ECM long enough to generate the force needed for cell movement but also be able to detach when necessary, allowing cells to stop and change direction.