Western Blot

Question 1

what does Western Blotting tell you?

Answer 1

• used to decipher the concentration of a specific protein in a sample
• determining the molecular weight of the proteins present in sample.

Question 2

What is densitometry?

Answer 2

It involves measuring the optical density of the bands provided by the results

Question 3

What does WB do vs ELISA?

Answer 3

1. WB separates proteins on a gel that can then be visualised
2. it binds denatured proteins
3. can determine the purity of the sample
4. more difficult experiment taking around 2/3 days to complete
5. it is good for determining the molecular weight of the sample
6. it is semi quantitative.

Question 4

What are the parts of the LAEMMLI sample buffer?

Answer 4

SDS, Glycerol, Bromophenol blue, and tris buffer

Question 5

Why are all the parts of the laemmli sample buffer present?

Answer 5

1. SDS: detergent that denatures proteins and coats with a uniform negative charge. This ensures that the proteins migrate according to their molecular weight and not charge.
2. Glycerol: to increase the density of the sample
3. Bromophenol blue: tracking dye
4. Tris buffer: maintains pH

Question 6

Step 1. of WB?

Answer 6

Gel preparation: stacking gel and resolving gel

Question 7

SDS-PAGE in WB?

Answer 7

This separates the proteins by denaturing them, this separates proteins based on size