Last week I performed Western blotting and I tried to separate a tight junction protein, zonulin, using Nitrocellulose membrane. But after performing the experiment I read that for separating low molecular weight proteins ( 100 kDa), we use PVDF membrane. Now I am wondering whether the use of nitrocellulose to separate zonulin made my experiment failure, as it's molecular weight is almost 200 kDa .
Anybody can help me?
The results and the image was not bad, but still I m concerned with the fact that for higher molecular weight proteins we need PVDF membrane.
Jun Li
Imam, it's unlikely to have anything to do with the membrane, though PVDF has a slightly higher binding capacity than nitrocellulose. Transferring proteins around 200kDa requires a little bit of SDS in your transfer buffer, and you might also need to increase your transfer time. Add 0.05% SDS to your transfer buffer, keep the methanol in. This should improve things. Good luck
Next time, I will add 0.05% SDS as per your suggestion and will let you know about the outcome. Thank you so much.
Neil A Bradbury
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