Lecture_6_notes_Protein_Techniques_BCH400_Spring_2025

Why is it important to purify proteins?

To study enzyme function, structural analysis, and determine post-translational modifications.

What are the general steps in protein purification?

Develop assay, choose source of protein, prepare tissue extract, protein fractionation, and determination of purity.

What does salting out involve in protein purification?

The process where proteins precipitate out of solution as salt concentration increases.

What is the purpose of dialysis in protein purification?

To remove excess salt or change the buffer in a protein solution.

What is gel-filtration chromatography used for?

To separate proteins based on size.

What moves through a gel-filtration column first?

Larger molecules, as they cannot enter the beads within the gel.

What is ion-exchange chromatography?

A technique where charged proteins are separated based on their net charge.

What differentiates anion exchange from cation exchange chromatography?

Anion exchange uses a positively charged column matrix while cation exchange uses a negatively charged column matrix.

What role does SDS play in SDS-PAGE?

SDS (sodium dodecyl sulfate) is a detergent that unfolds proteins and gives them a uniform negative charge.

What are the main components of immunoglobulin G (IgG)?

It is a Y-shaped hetero-tetramer consisting of heavy and light chains.

What is an epitope?

The part of an antigen that is recognized by an antibody.

What is the difference between monoclonal and polyclonal antibodies?

Monoclonal antibodies recognize a single epitope, while polyclonal antibodies recognize multiple epitopes.

What technique can be used to measure the activity of a protein?

Activity can be measured using assays such as the increase in absorbance at 340 nm when alcohol dehydrogenase catalyzes a reaction.

How is specific activity calculated?

Specific activity is calculated as total activity divided by total protein.

What does X-ray crystallography reveal?

It provides high-resolution structural information about proteins from crystal diffraction patterns.

What is 2-D nuclear magnetic resonance (NMR) used for?

To determine the structure of proteins in solution.

What is the purpose of Western blotting?

To identify specific proteins transferred from a gel to a membrane using antibodies.

What is the first step in HIV testing using ELISA?

To test for anti-HIV IgGs in the blood.

What is the significance of the specific activity measurement in proteins?

It assesses the purity and quality of the protein of interest.

What is the function of trypsin in protein cleavage?

Trypsin cleaves proteins at the carboxyl side of lysines and arginines.

What does SDS-PAGE separate proteins by?

It separates proteins according to size.

What can MALDI-TOF be used for?

To identify proteins based on the mass-to-charge ratio of peptide fragments.

What does affinity chromatography do?

It separates proteins based on their specific interactions with ligands.

What happens during isoelectric focusing?

Proteins are separated based on their isoelectric point (pI) in a gradient of pH.

How can peptides be sequenced using Edman degradation?

It progressively cleaves and identifies amino acid residues from the N-terminal end.

How are proteins detected on Western blots?

Using primary antibodies followed by detection with secondary antibodies or fluorescent reporters.

What is the role of antibodies in ELISA?

Antibodies are used to bind specific antigens for detection and quantification.

What happens if you run proteins through a high salt buffer in ion-exchange chromatography?

Proteins bound to the column may elute when salt concentration increases.

What is the function of 2-mercaptoethanol in SDS-PAGE?

It reduces disulfide bonds, allowing proteins to unfold.

How does protein mobility in electrophoresis differ?

It varies based on charge, size, and shape of the proteins.

What is Western blotting's primary purpose?

To detect the presence of specific proteins after gel electrophoresis.

What is the consequence of cleaving at different sites in polypeptide sequencing?

It determines the order of peptide sequences for accurate protein mapping.

How is the peptide map exercise relevant to protein sequencing?

It helps identify the order and structure based on specific enzyme digestion.

Why are monoclonal antibodies important in cancer therapy?

They are highly specific and can target cancer cells while leaving normal cells unharmed.

What does the specific structure of an IgG molecule facilitate?

It enables binding to pathogens and tagging them for immune response.

How can antibodies be produced for research?

By harvesting from hybridomas or directly from B-cell cultures.

What is a mature antibody fragment called?

Single-chain variable fragment (scFv), often used in therapeutic applications.