How to lower the background noise in the western blot membrane?

1、It could also be because  the blocking solution and the antibody have cross-reaction, it is recommended to replace the blocking solution or add Tween-20 to reduce cross-reaction.  

2、 It is recommended to shorten the exposure time, and you can try to manually process the film with developer and fixer, and check the results after pressing the photo for 1-2S.

This is a very common problem in western blot, and it can be caused by several factors:

(1)  Insufficient blocking. You may need to adjust the blocking conditions, including the blocking agent (milk or BSA), agent concentration, blocking time and temperature, etc.

(2)  Inappropriate primary antibody concentration or incubation temperature. This can be caused by a high concentration of the antibody. You need to set different dilutions of the primary antibody and determine the optimal antibody concentration for your target protein. The antibody incubation temperature (whether the membrane is incubated overnight at 4 degrees or several hours at room temperature) also makes a difference.

(3)  Poor washing steps. Check if the washing buffer is clean, fresh, and free of contamination. Repeated washing of 3-5 times and total washing of 15-30 min are necessary.

1, properly extend the blocking time

2, increase the number and time of washes

3, increase the dilution of secondary antibody

1. Try to reduce the amount of sample. 

2. Extend the closure time. Use BSA to block overnight at low temperature.

3. Increase the dilution of primary and secondary antibodies, and dilute the antibody with 5% skim milk powder.