Working with proteins (midterm 2)

Question 1

What is isoelectric focusing used for?

Answer 1

Determining pI

Question 2

How are proteins separated by pI in isoelectric focusing?

Answer 2

The gel contains a pH gradient. The sample has an electric current run through it and the proteins will migrate if they’re charged. Their charge changes as the pH changes, and will stop moving when their charge is 0

Question 3

Is the gel used in isoelectric focusing native or denaturing?

Answer 3

Native

Question 4

When would we use isoelectric focusing?

Answer 4

Testing the purity of a protein. Not very practical for separating out a protein from a solution. 2D electrophoresis

Question 5

What is 2D electrophoresis?

Answer 5

SDS-PAGE used along with isoelectric focusing to separate proteins by both pi and molecular weight

Question 6

How do you do 2D electrophoresis?

Answer 6

Run the protein solution through isoelectric focusing, then put that gel into an SDS-PAGE gel. Then run the SDS gel and look for the bands

Question 7

How do we know when a protein is pure?

Answer 7

Look for a single band by isoelectric focusing or SDS-PAGE, or when specific activity of an enzyme is no longer going up

Question 8

How do we get an integral membrane protein out of the membrane to study it?

Answer 8

Use a gentle detergent that will break apart the membrane. It will surround the hydrophobic portions of the protein with a micelle-like structure

Question 9

What is solubilization?

Answer 9

The disruption of the membrane with a detergent and forming the micelle structures around the hydrophobic portions of the protein

Question 10

What are two things that are problematic when doing protein purification?

Answer 10

Aggregation and proteases

Question 11

What type of proteins is aggregation a problem for when purifying?

Answer 11

Membrane proteins. The hydrophobic portions of the proteins aggregate together once they are out of the membrane

Question 12

How can we stop proteins from aggregating?

Answer 12

Detergents that will solubilize a protein but not denature it

Question 13

How can we stop proteases from messing up our protein purification?

Answer 13

Protease inhibitors or working fast at low temperatures

Question 14

What are 4 protease inhibitors?

Answer 14

PMSF, EDTA, Pepstatin, Leupeptin

Question 15

What is topology?

Answer 15

Localization of protein domains relative to the membrane

Question 16

How do we determine topology?

Answer 16

Treat the protein in the cell with a reagent that doesn’t cross the membrane. If the part of the protein that reacts is on the outside of the cell, we will see a reaction. If it’s on the inside, we get no reaction

Question 17

What type of reagents are used for determining topology?

Answer 17

Proteases, amine modifying chemicals, sulfhydryl modifying chemicals