Western Blotting/Immunoblotting Technique

Question 1

Immunoblotting/Western blotting

Answer 1

technique used to identify and even quantify the presence of a specific protein

Question 2

Step 1

Answer 2

Protein separated in polyacrylamide gel using electrophoresis

Question 3

Step 2

Answer 3

Transferred to nitrocellulose membrane (on positive electrode)

Question 4

Step 3

Answer 4

Coated with negative SDS travelled to positive electrode and captured by membrane

Question 5

Step 4

Answer 5

Inert protein used to coat membrane and primary antibodies added (only bind proteins of interest) at Fab regions

Question 6

Step 5

Answer 6

Then wash away unbound antibodies

Question 7

Step 6

Answer 7

Add secondary antibodies that bind to Fc domain of primary antibodies
linked to enzyme so location can be determined (cleavage=colour)