Chapter 3 - Protein Analysis

What are the 4 categories of protein separation techniques?

1. Size

2. Charge

3. Solubility

4. Specific binding affinity

What are the 4 separation techniques based on size?

1. Gel filtration chromatography

2. Dialysis

3. Ultracentrifugation

4. Electrophoresis

What are the 3 separation techniques based on charge?

1. Ion exchange chromatography

2. Electrophoresis

3. Isoelectric focusing

What is the separation technique based on solubility?

Selectively "salting out" of a solution

What is the separation technique based on specific binding ability?

Affinity chromatography

How does dialysis work?

Sample is place in a semipermeable membrane, small molecules diffuse out, large molecules are trapped

How does gel filtration chromatography work?

separate proteins based on size. Big proteins go fastest because the beads used in gel filtration have holes to trap small proteins

How does ion exchange chromatography work?

Negatively charged beads separate positively charged proteins on basis of charge density. Negative and neutral proteins wash through the column and are generally not bound

How does affinity chromatography work?

Separates proteins by shape(specific interaction). most specific. elution buffer used to wash out what sticks

How does electrophoresis work?

Charge in protein effects how it move in an electrical field, it moves bc of charge and size

How does ultracentrifugation work?

Separates molecules/particles on the basis of size, the unit is Svedberg (S) a sedimentation coefficient. S is dependent on mass, density, shape, and solution density

How does isoelectric focusing work?

A pH gradient is set up first. A mixture of molecules is applied, the electric field turns on and each protein moves to the position (pH) at which the net charge is zero

What happens when you remove a proteins from its native environment?

It changes the protein

What is the response to a foreign substance?

Specific antibodies (antigens) are generated

What is ELISA?

enzyme-linked immunosorbent assay

What does indirect ELISA detect?

antibodies

What does sandwich ELISA detect?

antigens

Where does cyanogen bromide cleave?

carboxyl side of methionine residues

Where does trypsin cleave?

at the carboxyl end of arginine and lysine

Where does chymotrypsin cleave?

at the carboxyl end of phenylalanine, tryptophan, methionine, leucine, and tyrosine

What are the steps of the Edman degradation?

Labeling —> release (so add onto the chain and then release) the peptide is shortened by one residue