Western Blot Optimization

[marcomonopoli]

Leave the gel in coomassie staining solution (500ml H20, 400ml methanol, 100ml acetic acid and 2.5g Brillant Blue) for 1 hour at room temperature, then remove the solution and leave the gel overnight in destaining solution (400ml methanol, 100ml acetic acid and 500 of water) at room temperature.
If the transfer did not succeed, on the following day you will see some proteins in the gel.
Hope this will help

[plummy]

I am working with Westren blotting to find the expression of eNOS, other colleagues from my department told me that eNOS is a very difficult protein to be detected. How can I manage this? On my membrane, the same sample with same concentration of the positive control (lung and kidney of mice) applied on different gels has shown bands with GAPDH, caveolin and COX-2, but no band on the gel to find eNOS. Can you help me to explain why? and how to optimise my result? My primary antibody was Polyclonal Rabbit Anti-eNOS/NOS Type III from BD Transduction. Both of the primary (1:1000) and secondary antibodies (1:1000) are dilute in skim milk 5% (TBS-Tween 20).
PS: I also work with cell culture (BAEC), in my 1st experiment to lysate the cells with RIPA, I can detect eNOS easily; however with the second time, I add orthovanadate into RIPA, I could not find the band for eNOS in Western blotting? I tried to find in literature, but orthovanadate could not be the cause for the problem. Would you please give me some comments? Thank you very much!

[sasha_028]

Instead of staining the gels, u can also stain the membrane with Ponceus Solution to check if most of the proteins have been transferred onto the membrane.