Hi,
I'm performing western blots on different samples and I'm facing a some problems after eletrotrasnfer with PVDF membranes.
In general, during the incubations with the primary and secondary antibodies prepared in 5% BSA in TBS-T and after the corresponding washes in TBS-T, it seems that the membrane starts to desintegrate. I'm attaching a sample image so you can understand what is the problem. The membrane is soaked in MeOH, trannferred to the transfer buffer for 15 min before sandwich assembly.
Does anybody knows what might be the problem?
Thanks in advance
I also observed something similar with my blots especially when I did semi-dry transfer. I did not have this problem with wet transfer, however. Do you already see such "patches" directly after the transfer? If you use semi-dry tranfer I would still add enough transfer buffer onto the blotting sandwich and the single parts of it prior to assembling. Also do not directly touch the membrane especially with your hands. Assemble the sandwich properly, so e.g. no overlapping.
Your transfer buffer contains MeOH, right?
Thank you Adrian Marc Hauri ! I only see these "patches" after blocking the membrane with 5% BSA and after incubation with antibodies. It seems that the membrane gets worse as I proceed with the incubation and washing steps with TBS-T.
I'm doing wet transfer and I assemble the sandwich inside a recipient with enough transfer buffer (containing 20% MeOH - Towbin buffer) that covers all the blotting parts. I'm also using gloves during all the process and the membranes in the image was cut in pieces so both parts are from the same original membrane.
After transfer, I'm air-drying the membrane and storing at 4 ºC for 2-3 days before re-wet in transfer buffer, wash in TBS and blocking as before. Do you think that it might be related to this air-dry process? Some transfer bufffer that might be attached to the membrane? Or perhpahs some BSA from the blocking buffer?
Thanks again!
Hello Luis
Such type of blots can appear due to A) overheated buffer. I hope you are maintaining the temperature of the buffer at 4°C during the transfer process. You can reduce the current and increase the transfer time to avoid overheating. You can keep the tank apparatus in ice during transfer. Another reason for such blots could be B) improper way of assembling the transfer sandwich that could increase the possibility of trapping air bubbles. I hope you must have moistened and fully saturated your filters and sponges with transfer buffer and used a glass rod or roller to remove the air bubbles between the gel and the membrane. C) Prewetting the membrane in methanol which has been done.
If everything above has been done properly then I will have a doubt on the air-dry process. Need to check that out.
Thank you.
Hello Luis Felix :)
Eu não sou muito esperta em WB, mas sempre que faço apenas activo a membrana PVDF aprox 1 min em MeOH antes de a colocar na sandwich. E depois sim fazer o transfer a 4 oC. Porque fazes este air-drying? Beijinhos
Dear Malcolm Nobre , thank you! I'm doing everything exactly as you describe (transfer overnight at low voltage and at at 4ºC, assembly of the sandwich on a recipient full of trasnfer buffer and assuring no air bubbles are present between the gel and the membrane, and prewetting the membrane in MeOH) and the only issue that I can find in the process is the air-dry process, which apparently would increase the retain of the proteins to the membrane. During the weekend I've tried without this process and I'm now incubating with primary antibodies to test.
Vera F. Monteiro-Cardoso hello!!! tudo bem? Obrigado. Esse processo é o que costumamos fazer aqui. No entanto, como muitas vezes deixo a transferir de sexta para sábado, quando venho retirar membrana deixo secar ao ar o que de acordo com a literatura deveria aumentar a retenção das proteínas na membrana. Após este processo, molho novamente membrana no tampão de transferência antes de continuar com processo de bloqueio e incubação com anticorpos. É a única coisa que me parece que possa influenciar estar a causar este efeito. De qualquer forma, estou a testar sem este processo a ver se a coisa vai. Beijinho!!
Hi Luis Félix
From what I see in the image I think you had problems in hydrate the membrane. Do you hidrate your membrane in water after the MeOH?
To prepare the PVDF membrane before the transfer we usually do 15sec in MeOH and then 1 min in water and then transfer buffer for at least 5 min.
Also, we always air-dry the membrane after the transfer and before the blocking and incubation with antibodies. However we always re-hydrate the membrane. So we incubate in ethanol 1 min, water 2min and TBS-T 5 min. And then blocking.
Hope it helps!
Hi Andreia N Carvalho , thanks for sharing your thoughts on this! It seems that if I do not air-dry the membrane before blocking, the all process goes well and the membrane is not damage at the end. Perhaphs there is some problem (dust or other particles) that remains in the membrane and roriginates these spots in the membrane.
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