SDS PAGE and WB

Question 0

What is poly acrylamide

Answer 0

Polymer of acrylamide monomers. Thermostable, transparent, strong, chemically inert.
Polymerizes into long chains in presence of free radicals (ammonium persulphate)
Xlinked in presence of N,N-methylenebisacrylamide

Question 1

What is SDS in SDS-PAGE

Answer 1

A detergent. Negatively charge sulphate attached. Denatures proteins and covers with negative charge. Result - Linear primary structure proteins with neg charge.

(1.4g SDS bing 1g protein)

Question 2

Why store acrylamide in cool, dark and dry place

Answer 2

In water autopolymerisation of acrylamide. Also prevents hydrolysis.

Question 3

What inhibits polymerization

Answer 3

Oxygen

Question 4

Why be careful when handling acrylamide

Answer 4

Neurotoxin!

Question 5

What stabilizes polymerization and x linking

Answer 5

TEMED

Question 6

Difs between two types of gel in SDS-PAGE

Answer 6

Stacking - large pore, lower pH (6.8), concentrates proteins in single sharp band
Resolving - small pore, higher pH (8.8), proteins separate according to size.

Question 7

What is used to help gel set

Answer 7

Butanol/ isopropanol

Question 8

Role of buffer in SDS-PAGE

Answer 8

Constant pH

Ions to support conductivity

Question 9

Common buffer for SDS-PAGE

Answer 9

Tris-glycine

Question 10

Thre NB current carrying ions in SDS-PAGE

Answer 10

Glycine
Chloride ions
SDS/proteins

Question 11

Buffer used within SDS-PAGE gel

Answer 11

Tris-Cl

Question 12

What does a discontinuous buffer system mean

Answer 12

Buffer in the gel and in the tank are different

Question 13

What’s the purpose of discontinuous buffer system

Answer 13

Cl ions migrate faster than Gly, (low pH in stacking gel favours zwitterionic form of Gly) a region of low conductivity and high voltage drop formed, therefore proteins stack. Increase in pH of resolving gel ionizes Gly therefore Gly runs faster and proteins unstack and separate.
Advantage - increase in resolution of bands

Question 14

Three purposes of loading buffer

Answer 14

1. Glycerol increases density of buffer. Protein loads evenly
2. Adds colour to sample. Helps with loading and tracking of progress (bromophenol blue - tracking dye. Small, runs faster than most proteins.
3. Beta-mercaptoethanol - protein denaturation

Question 15

How does B-mercaptoethanol work

Answer 15

Besides SDS, briefly heating protein with BME allows reduction of disulfide linkages therefore preventing tertiary and quaternary structures.

Question 16

How does coomassie brilliant blue work

Answer 16

Anionic dye. Exposed hydrophobic regions of protein bind to CBB. Usually used in methanolic and acetic acid(fixes proteins to gel) solution.

Question 17

Uses of SDS-PAGE

Answer 17

Est protein size
Protein identification
Determining sample purity
Quantifying proteins
Western blot apps

Question 18

Function of western blot

Answer 18

Identify specific protein
Determine MW of protein
Measure relative amount of protein

Question 19

Describe transfer of proteins in WB

Answer 19

To make proteins accessible to Ab detection, transferred onto nitrocellulose membrane. Use electric current

Question 20

Why is membrane blocked in WB

Answer 20

Prevents non-specific binding of Abs to membrane.. (BSA or non-fat dry milk)

Question 21

What is 2ndry Ab usually linked to

Answer 21

An enzyme eg. Horseradish peroxidase or alkaline phosphatase

Question 22

What is the purpose of loading controls in WB

Answer 22

Often called housekeeping genes. Confirms that protein loading is same in all lanes. NB when comparing expression levels between separate samples.

Usually B-actin, COX-4, GAPDH or tubulin

Question 23

Why must one be careful when preparing SDS-PAGE gel

Answer 23

Acrylamide is very toxic. Do not allow contact to skin, wash areas of contact, DO NOT pour unpolymerized gel down sink. Dispose polymerized gel in solid waste.

Ammonium persulphate - use fresh stock, goes off quickly, dilute and pour down sink.

TEMED - harmful if inhaled / ingested. Store in dark glass.