Protein Detection & Interaction Techniques: Western Blots, Immunofluorescence, Co-IP, EMSA, and CRISPR Mutagenesis

What is the purpose of untargeted protein experiments?

To provide a global overview of all proteins in a sample without identifying specific proteins.

What methods are used in untargeted protein experiments?

SDS-PAGE and 2D-PAGE.

What is the main limitation of untargeted protein experiments?

They do not identify specific proteins and only show total protein patterns.

What is the goal of targeted protein experiments?

To detect one specific protein out of a complex mixture using antibodies.

What technique is used to detect the abundance and approximate molecular weight of a specific protein?

Western blot.

What does immunofluorescence detect?

The location of a protein in a tissue or cell.

What is the purpose of immunogold labeling?

To detect subcellular localization at very high resolution.

What are the steps involved in performing a Western blot?

1. Denature protein mixture, 2. Run SDS-PAGE, 3. Transfer proteins to membrane, 4. Block, 5. Wash, 6. Incubate with primary antibody, 7. Wash, 8. Incubate with secondary antibody, 9. Add substrate, 10. Detect signal.

What information does a Western blot provide?

Protein abundance and molecular weight.

What is a major limitation of Western blotting?

It provides no spatial information and cannot recover proteins.

What does immunofluorescence provide that Western blotting does not?

Exact location of the protein within tissues or organelles.

What is a control for primary antibody specificity in protein detection?

Using a negative control sample, such as a mutant lacking the protein of interest.

What is a control for secondary antibody specificity?

Omitting the primary antibody to check for nonspecific binding.

What is the difference between transcriptional and translational fusions?

Transcriptional fusions contain only the promoter; translational fusions include the promoter and the entire open reading frame (ORF) of the gene.

What does a transcriptional fusion tell you?

Where the promoter is active, but not where the actual protein localizes.

What does a translational fusion allow you to visualize?

The actual protein's localization, movement, and sometimes function.

What research question can be answered using translational fusion experiments?

Where is the protein localized?

What is one control for translational fusion experiments?

Using a no-primary antibody control to test background fluorescence.

What can you monitor with translational fusion experiments after treatment or mutations?

Hormone responses, stress responses, and changes in protein localization.

What is the significance of a fusion protein rescuing a mutant phenotype?

It indicates that the fusion protein is functional.

What is a limitation of immunofluorescence and immunogold techniques?

They can be time-consuming and may not detect low-abundance proteins.

What is the role of a secondary antibody in targeted protein detection?

To recognize the constant region of the primary antibody and usually has a conjugated tag.

What is a common method for detecting signals in Western blotting?

Chemiluminescence, fluorescence, or colorimetric output.

What does a pre-stained ladder confirm in Western blotting?

That proteins have been transferred properly.

What is the purpose of probing a duplicate blot with a housekeeping protein?

To show equal loading across samples.

What is the importance of using a mutant control in antibody specificity?

To ensure the primary antibody does not bind to unrelated proteins.

What is the significance of using a fluorophore in antibody detection?

It allows for visualization of the protein location through fluorescence.

What is functional complementation in protein analysis?

Transforming a mutant lacking a protein to see if a fusion can rescue the phenotype.

How can you confirm that a reporter is attached and not cleaved off?

Using SDS-PAGE or western blot with an antibody against the reporter (e.g., anti-GFP) to confirm the fusion protein's size.

Why is it important to ensure observed localization is due to the protein and not free GFP?

Because GFP alone diffuses everywhere, making it necessary to show a distinct localization pattern.

What is the purpose of a negative control in a translational fusion experiment?

To demonstrate that localization is due to the YFG sequence, not promoter activity differences.

What is the difference between native and constitutive promoter constructs in overexpression control?

Native promoters reflect natural expression levels, while constitutive promoters drive constant expression regardless of conditions.

What is one benefit of using translational fusions?

They allow highly specific protein detection with no cross-reactivity with similar isoforms.

What is a limitation of translational fusions regarding organism transformation?

Some organisms are hard to transform, necessitating the use of model systems.

What can cause false localization in fusion proteins?

Proteolysis may separate the reporter (e.g., GFP) from the protein.

Why can proteomics be more challenging than genomics or transcriptome profiling?

Proteins degrade easily, are less stable than DNA or RNA, and have more chemical diversity due to 20 amino acids.

What are post-translational modifications (PTMs) and why are they significant?

PTMs like phosphorylation and glycosylation change mass, charge, or conformation, complicating detection.

What is the relevance of studying protein-protein interactions?

They are fundamental to biological processes, controlling signaling pathways, transcriptional regulation, and metabolism.

What is the first step in a pull-down assay?

Attach the bait protein to beads, often using a tag like GST or FLAG.

What happens during the washing step of a pull-down assay?

Nonspecific binders are removed to ensure only specific interactions remain.

What is the purpose of analyzing proteins by SDS-PAGE after a pull-down assay?

To detect physical interactions between the bait protein and unknown prey proteins.

What is the first step in a co-immunoprecipitation (co-IP) experiment?

Prepare a cell or tissue lysate.

How does co-IP differ from pull-down assays?

Co-IP uses antibodies to capture protein complexes, while pull-down assays use tagged bait proteins.

What is one benefit of co-immunoprecipitation?

It allows for the study of protein interactions in their native context within cells.

What is a limitation of pull-down assays?

They may miss transient or weak interactions that are not captured during the binding process.

What is the significance of using different colored reporters in translational fusions?

They allow for the co-localization of multiple proteins to observe interactions.

What is a common method for analyzing eluted proteins from a pull-down assay?

SDS-PAGE, followed by staining methods like Coomassie or silver stain.

What is the role of secondary antibodies in co-IP experiments?

They bind to the primary antibody, allowing the antibody-protein complexes to stick to the beads for isolation.

What is the purpose of negative controls in pull-down assays?

To detect nonspecific binding and ensure the antibody only pulls down real complexes.

What is a positive control in a pull-down assay?

An overexpressed prey protein that helps detect weak or transient interactions.

What are the benefits of a pull-down assay?

Fast, easy, inexpensive, good for stable interactions, and can detect interactions using tagged proteins.

What are the limitations of pull-down assays?

Requires strong interactions, may miss weak interactions, can cause artifacts, and bait may block important binding sites.

What is a key benefit of Co-IP (Co-immunoprecipitation)?

Proteins remain in their native conformation, capturing interactions as they occur in vivo.

What is a limitation of Co-IP?

Requires a high-quality specific antibody and may cause nonspecific interactions due to extract preparation.

What is the first step in a gel-shift assay (EMSA)?

Isolate the DNA fragment containing the putative cis-element, usually done using PCR.

Why is DNA labeled in a gel-shift assay?

Labeling provides high sensitivity since DNA-binding proteins are often low in abundance.

What is the purpose of running samples on a non-denaturing polyacrylamide gel?

To preserve protein-DNA interactions.

What does a shift upward in a gel-shift assay indicate?

Protein-DNA binding.

What is the role of a specific competitor in control scenarios of a gel-shift assay?

It competes for the protein, indicating whether binding is specific or non-specific.

What happens when random DNA is added as a non-specific competitor?

If binding is specific, the shifted band remains unchanged; if non-specific, the shift decreases with added random DNA.

What is the first step in using CRISPR for site-directed mutagenesis?

Fuse crRNA and tracrRNA to create sgRNA (gRNA).

What is the function of the PAM sequence in CRISPR?

It is required for Cas9 to cut the DNA.

What are the two pathways for DNA repair after a double-stranded break (DSB) in CRISPR?

NHEJ (Non-Homologous End Joining) and HDR (Homology Directed Repair).

What type of mutations does NHEJ typically create?

Random mutations, often resulting in INDELs (insertions/deletions).

What is the purpose of HDR in CRISPR?

To introduce precise, template-guided mutations using a donor DNA template.

What is a no-gRNA control in CRISPR experiments?

It ensures that Cas9 does not cut DNA in the absence of gRNA.

What are the benefits of using CRISPR for genome editing?

Fast, least expensive, highly programmable, works in many organisms, and can generate both knockouts and knock-ins.

What is a limitation of CRISPR technology?

Requires a PAM sequence, can have off-target effects, and HDR efficiency is low in many organisms.

What is the significance of using a donor DNA template in HDR?

It allows for site-specific, precise genome editing by copying the donor sequence during repair.

What is the outcome of using a scrambled gRNA in CRISPR experiments?

It tests for off-target effects of CRISPR components.

What is a supershift control in a gel-shift assay?

Adding an antibody specific to the protein causes a slower, 'supershifted' band, proving the identity of the DNA-binding protein.

What is a key advantage of the gel-shift assay (EMSA)?

It can detect multiple proteins binding to the same DNA fragment.

What is a limitation of the gel-shift assay?

Cannot recover the DNA-protein complex from the gel due to low amounts and reliance on autoradiography.

What type of interactions may be missed in pull-down assays?

Weak or transient interactions.

What is the effect of overexpression in pull-down assays?

It may cause artifacts that affect the results.

What does a gel-shift assay reveal about protein-DNA interactions?

It shows the mobility differences between free DNA and bound DNA.