Hello,
I have performed two westerns in parallel, one using fresh buffer (made with old 10X tris-glycine) and the SDS gel was made 4 days prior to western; and the other western was performed using old transfer buffer (made with newer 10X tris-glycine) but the SDS was made on the same day.
As you can see in the image, the one with fresh buffer had a very incomplete transfer while the one with old transfer buffer is transfered better.
Any thoughts on what could the the reason for this poor transfer? Could it be the old SDS gel, or the transfer buffer itself that had the been prepared with old stock solution of tris-glycine?
Thank you,
Ladan
Can I help?
I am looking at your result,but what I Don't see if the fresh buffer is better or not. The new transfer buffer is far better than the old transfer buffer. (Do you mean the New Tris glycine buffer?)
It is always best to check the conditions of all the stocks and materials used including storage conditions. How long would you take to make a membrane SDS, and complete the western blot? It is not bad four days. Maybe suggest it depends on the protocol. How many reuses can you make to a transfer buffer? Does it depend on the original protein? (Conditions like Coomasie blue...? ) I Don't think the different stock on 10x tris glycine makes a drastic difference.
Can we receive feedback? I would like to know what happened afterwards. I do appreciate if you could clarify the New or old transfer buffer.
Thank you.
I think you scrambled your situations! Because the gel on the left has transferred very well with new buffer as you said in the pic, in contrast, the one in the right looks bad because little amount of protein can be seen. Have in mind that, when you perform protein transfer, the buffer is recommended to be new or to have few uses (I only use it two times, even a 3rd time, beyond that is not suggested). When you start to reuse the transfer buffer 1X, the buffer losses methanol (a fraction evaporates with each use), which significantly decreases the overall efficiency of a transfer procedure. So, try to perform protein transfer with fresh buffer or one with few uses and store it at 4°C to ensure methanol "integrity". If you see any precipitates (because of filter paper) it's better to filtrate it.
Hope it helps!
Lady López Thank you for your response. I am unclear of how you say the gel on right has poorly transferred. I should have been more clear. These are the SDS gels which are Coomassie-stained such that we can visualize how complete the transfer was. As we can see, the one on the LEFT still holds a good amount of protein within the SDS gel and all these bands on this gel have incompletely been transferred onto the membrane during the western. Nevertheless, I agree about what you said regarding methanol evaporation.
@Ladan Kalani Sorry, I totally misread the question. Well I am kind of puzzled because it should be backwards right? So my question is, was this a wet transfer or a semi-dry one? And regarding the buffers, I think the fresh stock (the one in the left) wasn't well prepared because it has nothing to do with the gel, I have prepared gels days earlier and as long both the SDS-PAGE buffer and the transfer buffer are okay, the run goes well!
Lady López it is indeed unexpected to have worse transfer using fresh transfer buffer; this was transfer onto a nitrocellulose membrane so I did not prewet in methanol. I agree with you that the problem is with the stock solution of tris-glycine with which the fresh buffer was made! Thank you for your clarification!
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