Hi,
I am facing this problem sometimes with PAGE transfer. After I stained with Poncuaue I saw some white horizontal lines on my membrane which are consistent with my sandwich holes. I can understand the air bubbles but I don't know why these holes came up sometimes. Any suggestions? What is the reason behind this? Thanks in advance.
Were they transferred using the wet or semi-dry method?
Did you use the roller to press the gel-membrane-filter sandwich?
What about the pre-wetting step of the membrane? If you are using PVDF then you have to activate in methanol/transfer buffer and should not be dried out.
From the ponceau image of both membranes, one can observe that the small proteins are hardly giving any clear bands as compared to the upper half of the membrane.
What percentage and running conditions of the gel are while transferring?
Try to soak your membrane in transfer buffer for at least 5 minute minutes to remove SDS
Vikrant Mehta Thank you for answering back and happy new year.
I used wet method
Yes I have used roller
I activated PVDF membrane for 2-3 mins in methanol
Both are %10 gel
Thanks for this advice I will soak in transfer membrane.
I am guessing that our filter sandwich sponges were not washed properly. It may have contained too much salt in it that's why I may face this kind of problem. I will wash them before I use in each time.
Thank you for your reply.
Adil Vural Happy new year to you as well. So I was just wondering about the low efficiency of the transfer of small mol wt proteins. Any reason for that?
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