Good day, is the transfer buffer used for western blot re-usable for other occasions? I use 20% methanol and 10% 1X Tris Glycine SDS (TGS) buffer. I ask because I wish to not waste methanol in preparing the buffer. If so, I can store it at -4C fridge until another western blot experiment is present? The type of western blot used in our laboratory is wet and not semi-dry.
Thank you.
hey, I have reused the transfer buffer in the past with a maximum of 3 runs but I stopped doing so because I became worried about buffer capacity. In all honesty, I would not resuse the transfer buffer. I am sure that your time and samples are more previous and valuable than saving a few dollars by making up fresh buffer. Like all buffered systems, there is a point when the buffer capacity has been exceeded. I am not exactly sure when this is but it would not be ideal to run valuable samples and have the transfer go bad. If you are adverse to using so much methanol, you could always try semi-dry even though you stated that your lab performs wet transfers. You could also use Biorad's new turbo blot system which uses ethanol and not methanol.
Hi,
Yes offcourse you can reuse your transfer buffer like 1-2 times but make sure that after the 1st time keep it in 4 degrees and as the methanol level diminishes the effectiveness decline too.If you need a good result and your protein is precious to you avoid using reuse because you might get unreliable result.
I hope this will help you.
All the best!
I believe using the buffer two times should be fine. We do that always in the lab and the blots look fine.
Hi Eduardo! I'm in a lab in Argentina and we reuse the transfer buffer several times because methanol and ethanol are too expensive here. We have never had any problems. We just make sure that we do a Ponceau Red staining in order to check if the transfer went OK.
I would not re-use transfer buffer, simply because it will become contaminated with protein and perhaps your protein of interest which could increase background. Remember that the membrane will more than likely not bind all protein present on the gel therefore some will be present in the buffer which is not what you want.
If you are worried about methanol, try leaving methanol out and comparing transfer efficiency. A lot of proteins do not require methanol for efficient transfer. One of the prime reasons for using methanol is in the transfer of more hydrophobic proteins. All our transfer buffers used in the lab do not contain methanol. If using PVDF membranes we do use a small amount of methanol to wet the membrane to allow for efficient equilibration in transfer buffer. We make sure that we incubate our gels and membranes at least 15 minutes in transfer buffer prior to transfer. There are many buffers for transfer of proteins, many without methanol, try BioRad website for info booklet. Experimenting with different buffers is the only way..thats science!!
Yes you can, I re - use the transfer buffer for 3 times and I do not have issues with my transfer. I make sure I keep it at 4 C after using it for the first time and its fine to reuse. I know some labs would also add a bit of methanol to it after using it for several times to aid maintain its effectiveness.
Have you considered to use Dunn's buffer (10 mM NaHCO3 and 3 mM Na2CO3 with 50 µM SDS for large proteins, doi:10.1016/0003-2697(86)90207-1) instead of Towbin's? It is not only more effective but also so cheap that reuse is unnecessary. And you produce no organic waste that makes disposal problems.
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