I'm a new bird in doing western blotting. I'm now doing a western blotting for the membrane protein on culture cell. After immunostaining with my primary antibody and secondary antibody, the positive signals were visualized by using ECL+ and expose for 3 minutes. Eventually, I found that there are lots quite a number of no of non-specfic bands and their abundance increase with time. I don't what the problems. I here quote the procedure for my washing and hope there is somme comment on that:
Step 1.Wash twice (5 minutes each) with Washing Buffer A
Step 2.Block the blot for 2 hours at room temperature with Blocking Buffer
Step 3.The blots to be incubated overnight at room temperature with the same blocking solution containing 3mg/mL of 1˘X antibody.
Step 4.The blot to be then washed 5 times (5 minutes each) with Blocking Buffer.
Step 5.Then, incubate the 2˘X antibody of 1:5000-fold dilution in Blocking Buffer for 1 hour at room temperature.
Step 6.Then, the blots to be washed in twice (5 minutes each) with Washing Buffer A.
Step 7.Then, the blots to be washed with Washing Buffer B twice; 5 minutes and then followed by 10 minutes.
Step 8.The blots to be washed with Washing Buffer A for 5 minutes
Step 9.Then, the blots to be washed with Washing Buffer B for 10 minutes
Step 10.Finally, the blots to be washed with Washing Buffer A only.