This is regarding the ability of PVDF membrane to immobilize proteins onto it. If I run the transfer for prolonged periods, will the proteins stick onto the membrane or do they pass through the membrane and get lost in the buffer?
Suhas,
PVDF membranes are quite strong and hence proteins dont pass through them. If you are looking for high molecular weight proteins (> 200 kd) you can transfer for prolonged periods. But make sure you put low voltage and at 4 C. Hope this helps!
Mr.Angara, Thank you for your valuable suggestion. However the proteins of my interest are in the range of 30kDa and 50kDa. I had to standardize a new Western Transfer System from Biorad. Transfer was kept at 25V, 90mA for about 3 hrs. The protein transfer onto the PVDF membrane was not very evident from Ponceau staining, and I did not see proteins left over in the gel too. Hence I had the above doubt. Thank you sir.
Preincubation of the gel in the transfer buffer for 15-20 min facilitates the proteins transfer to the membrane. It normally works well with SDS PAGE separated proteins.
As Dr. Angara wrote with high molecular weight protein there are usually few problems during transfer. But if you need to transfer low molecular weight proteins then be careful on voltage setting and time of transfer. I usually use over night transfer at 4°C without problems, at least for proteins in the range 40 - 200 kd.
Suhas,
I think you should load more protein sample into the gel. May be your model systems don't have enough protein of interest in them. First, optimize your transfer system using a loading control protein like B-actin or GAPDH. If this works with your new system, then we can figure out the transfer conditions for your protein of interest.
Hi Suhas,
Ponceau staining does not work very well with PVDF membranes. So you actually might have a lot of protein there but don't detect it very well. either stain it with coomassie (just very briefly; 5min or so) or, if you want to stick to the ponceau, try a nitrocellulose membrane where the ponceau works much better.
Are you using the BioRad Wet Transfer System or Semidry? If you are doing wet western blotting I used to get a good transfer by blotting for 1h at 100V (I was looking for proteins in a range of 50-80KDa).
Hope that helps a little bit.
Mr. Maurizio Gualtieri, Thank you for your inputs.I am using a wet transfer system from Biorad . Proteins of the range of 40-200kDa is also a part of my work. For this, could you please tell me the exact voltage and mAmp settings (I will standardize it later based on my results) for transfers at 4 degrees overnight ? Kindly advise . Thank you.
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