We are currently facing a problem regarding the Western blot technique. Proteins above 70 kda don't seem to be transferred to the membrane. So far we have ruled out: The system, the samples, all the reagents used in the making of the gel and transference (including the buffer). When we stain the gel with blue comassie we can see all the proteins there, this led us to think that the problem is in the transference. Even though the ladder transfers well, the proteins do not appear in the top part of the membrane when we dye with the ponceau. Has anyone faced the same problem? We have tried everything!
Thanks in advance.
This membrane is from a 10% gel, notice the weak signal in proteins above 70kda.
You are right, the problem is in the transfer procedure. The fact that the higher MW proteins of the ladder were not transferred very well might indicate that you need to increase the time of the transfer (we do it for 2 hours at 100 V constant voltage in wet/liquid western blot procedure in the cold room). If you run dry or semi/dry western blot technique I would also recommend to switch to wet/liquid old procedure.
Vyktor, thanks for answering.
We do it for 2:30h at 100v in the cold room and we use the wet procedure
In my opinion you should try adding 0.1% SDS to the Transfer Buffer to ease proteins' transfer. For the tra transfer procedure I usually use 90 minutes at 250 mA.
Hope this could help!
Hi
Please have a look on this page. Its explains very well.
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6148.pdf
Hope I colud help.
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