Lab 5 - Gel Electrophoresis and Molecular Weight Estimation

Question 1

What is the purpose of gel electrophoresis?

Answer 1

To estimate the protein molecular weight by denaturing polyacrylamide electrophoresis, creating decreased pores to separate the proteins.

Question 2

What properties did we use to separate proteins in the gel electrophoresis experiment?

Answer 2

Size

Question 3

How to prepare the gel electrophoresis sample

Answer 3

1. Centrifuge 1.5 ml of Tetrahymena and decant supernatant into the sink
2. Mix Tris-HCl with a pH = 8.0, SDS, glycerol, and 10% Beta-mercaptoethanol (BME) and heat solution for 5 min at 100 degrees celcius
3. Add 100 micro liters of sample buffer to the solution, mix and heat at 95 degrees celcius for 5 min
4. Add 10 micro liters Tracking dye to each sample

Question 4

Purpose of Tracking Dye

Answer 4

BPB: Indicates when the gel has run to completion

Question 5

Purpose of SDS

Answer 5

Increase density of samples

Question 6

Purpose of BME

Answer 6

Reducing Agent

Question 7

Purpose of glycerol

Answer 7

Increase density of samples

Question 8

Purpose of heating the sample

Answer 8

Solubilize the proteins, reduce disulfide bonds, allows SDS to bind to all proteins

Question 9

What is a denaturing or reducing gel?

Answer 9

It separates proteins based only on their molecular weight because all proteins carry a negative charge from the SDS added

Question 10

Stacking Gel

Answer 10

Added after the separating/resolving gel has polymerized; found on the top of the gel; consists of 4.0% polyacrylamide and separates proteins by a gross association of large versus small proteins

Question 11

Resolving Gel

Answer 11

Is added first; found on the bottom of the gel; consists of 12.5% polyacrylamide and separates proteins based on molecular weight

Question 12

Separates proteins based on molecular weight

Answer 12

Resolving gel

Question 13

Separates proteins based on association of large vs samll

Answer 13

Stacking gel

Question 14

Graph a ladder

Answer 14

x-axis = Distance migrated (cm)
y-axis = 10^-3 Molecular Weight

Question 15

How to determine unknown properties

Answer 15

Compare bands to known ladder

Question 16

Purpose of 2-mercaptoethanol

Answer 16

Reduce and destroy the disulfide bonds creating single polypeptide bonds

Question 17

How does separation of proteins change when acrylamide percentage changes?

Answer 17

The smaller the known weight, the higher the percentage that should be used