I have been doing western blot for STAT1 gene. I have tried primary antibody from different companies Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb #9167 ( Cell Signaling Technology),Phospho-STAT1 (Tyr701) Polyclonal Antibody ( Invitrogen), proteintech as well. Until now I have not seen any band after trying several time. Can someone please suggest me why I am not getting expected band for STAT1 gene. I am getting for beta actin which is internal control, Unfortunately no band seen for STAT1 gene.
Hi Ranjit Kumar Das
You need to take care about the type of blocking solution, if you use skim fat milk, maybe it is the problem. So, you can try another blocking one.
Another possible cause (Although I think it is not the case here) is the ability of your S.Ab to bind the P.Ab. So, to roll out this, you can plot a drop of P.Ab directly on the membrane, wait to dry, then add a drop of the S.Ab over it and another one straight on the membrane, then proceed normally with other WB steps. If there is a light in the both dots, then there is no problem in your antibodies and you have to seek another cause. If there is only a light in the S.Ab dot, then there is a problem in the binding between your antibodies. If there is no light in both dots, then the problem is the S.Ab.
Hope this was helpful.
Hi Mohamed Khashan Thank you so much for your clarification. Yes, I am using 5% non fat dry milk to block the membrane. That is also could be the reason. If you do not mind can you please review my western blot protocol. Since I am the first one in our lab who is doing western blot. We do not have any established protocol. I would be happy if you could review my western blot protocol. Thanks
With anti-phosphoprotein-antibodies, definitely avoid milk based solutions for blocking and dilution purposes, as the phosphates in casein will catch your antibodies.
As an alternative, BSA at 1..5% m/v usually works well. If you still get no signal, replace phosphate based buffers with tris based solutions.
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