Proteomics

What is the proteome?

All proteins in a cell encoded by the genome.

What is proteomics?

The study of proteins and their activities.

What methods can be used to break cells for protein extraction?

Manual methods (blender, sonicator, glass beads), detergents to remove membranes, or freeze/thaw cycles.

How can proteins be separated?

By size and/or charge using polyacrylamide gels and column chromatography.

Why do amino acids not have the same charge?

Because they have different R groups.

What role does SDS play in protein analysis?

SDS denatures proteins and gives them a net negative charge.

How can specific proteins be identified after gel separation?

Using antibodies and western blots to locate proteins on the blot.

What is the purpose of a Western blot?

To identify specific proteins and determine their presence in a sample.

How does an antibody recognize a protein?

Antibodies bind to specific epitopes on proteins.

What is mass spectroscopy used for in proteomics?

To measure the molecular weight of proteins, allowing identification of the proteins.

What are some common protein tags used in proteomics?

MBP, His-tag, FLAG-tag, and Strep.

What is the benefit of adding protein tags?

They allow for easy isolation of specific proteins.

What is the principle behind phage display?

To screen for particular proteins based on their function.

What can Yeast 2-Hybrid method identify?

Interactions between proteins.

How does the Yeast 2-Hybrid method work?

Two proteins interact to form a transcription factor that activates a reporter gene.

What is the role of the reporter gene in Yeast 2-Hybrid?

To indicate whether the interacting proteins are present.

How can the results of Yeast 2-Hybrid be confirmed?

By running additional parallel immunoprecipitations.

What is co-immunoprecipitation (co-IP)?

A method to isolate protein complexes by using specific antibodies.

How does immunoprecipitation work?

An antibody binds to its epitope on a protein, allowing isolation of the complex.

What is a precipitate in the context of co-IP?

A heavy complex formed by antibody-protein interactions that can be isolated by centrifugation.

What happens to proteins interacting with the target protein during co-IP?

They also get precipitated along with the target protein.

How can the presence of interacting proteins in co-IP be verified?

By checking if the other proteins are also present in the precipitate.

What is the significance of mass spectroscopy like MALDI-TOF?

It allows for precise measurement of proteins' molecular weight.

Why is understanding each protein's molecular weight important?

It helps in determining the identity of the protein.

What type of chromatography is commonly used for protein separation?

Column chromatography.

What technique is analogous to Western blots for DNA?

Southern blots.

What is the function of the activator domain in Yeast 2-Hybrid?

To activate the transcription of the reporter gene when proteins interact.

What is the process of maturing yeast strains in Yeast 2-Hybrid?

Mating strains carrying two separate vectors to create cells with both vectors.

Why are protein tags useful when cloning coding sequences?

They allow for easier isolation and identification of the protein of interest.

What is the advantage of using detergents in cell lysis?

They help remove cell membranes for easier protein extraction.

Why is SDS important in polyacrylamide gel electrophoresis?

It standardizes the charge of proteins for separation based on size.

Can proteins have a positive charge in a gel?

Generally, proteins have a negative charge when treated with SDS.

What does the term 'epitope' refer to?

The specific part of the antigen that an antibody recognizes and binds to.

What does MALDI-TOF stand for?

Matrix-Assisted Laser Desorption/Ionization Time of Flight.

What type of interactions can phage display analyze?

Protein-protein and protein-RNA interactions.

What is the primary purpose of a reporter gene in biological assays?

To measure gene expression or the activation of a specific pathway.

What is a key requirement for proteins to be identified using co-IP?

They must be part of the same complex or interact under specific conditions.

What differentiates mass spectrometry techniques?

Different methods like MALDI-TOF and electrospray offer various detection and analysis capabilities.