Answer to Question #61040 in Biochemistry for Mia

Question

I have been working on western blots for about a month now using actin and fetuin-b primary antibodies to no avail. My background is always high and I only had one good result. I want to know if there is a certain blocking buffer I should use instead of 5% milk with my membranes to get a better result.

Answer 1

Answer: For western blotting you may use as well 5% non-fet milk as 3% bovine serum albumin (BSA). For better results and decreasing background I recommend you proper washing your membranes with TBST or PBST buffer 4-5 times for 5 min after primary and secondary antibodies. Also you must use blocking buffer (milk or BSA) for at least 1 hour (room temperature) or overnight (+40C). Some protocols I work with recommended to use primary and secondary antibodies diluted in blocking (milk) buffer.