Western Blotting Method

Question 1

What is western blotting method

Answer 1

Widely used to identification. Detection, and quantification of specific proteins

Question 2

Western blotting method steps

Answer 2

1sample preoaration
2protein quantification
3gel preparation
4sample and ladder loading
5Electrophoresis
6protein transfer
7blocking
8primary antibody incubation
9washing
10secondary antibody incubation
11washing
12detection
13data analysis

Question 3

Sample preparation

Answer 3

Cell or tissue lysis to extract protein. And also added protease to prevent protein degradation

Question 4

Protein quntification

Answer 4

Measure protein from Bradford to ensure equal amount of proteins are loaded in to each well

Question 5

Gell prelaration

Answer 5

Prepare and assemble the poly acramydegel.
SDS- denature proteins give negative charge

Question 6

Electrophoresis

Answer 6

Run the gel under on electrical field

Question 7

Protein transfer

Answer 7

Transfer protein in to nitro cellulose paper

Question 8

Blocking

Answer 8

Block non specific binding sites

Question 9

Washing

Answer 9

Washing with TBST to remove unbound primary antibody

Question 10

Secondary antibody incubation

Answer 10

It bind with primary antibody. This antibody link with enzyme

Question 13

Washing 2

Answer 13

Remove excess secondary antibody

Question 14

Detection

Answer 14

Add a substrate ECL that react with enzyme to produce light

Question 15

Data analysis

Answer 15

Analyse the band compare with ladder that means with molecular weight marker target protein compare