7 replies to this topic

[globy123]

Hi. I was doing a Western blot today and I accidentally used Transfer buffer when I was running the gel instead of using Running buffer. Will that ruin the gel? Should I start over?

[jes]

i guess it will ruin the gel. or it simply won't run. both buffers are made up of different consituents.

[Luthien]

Well, if your transfer buffer is with SDS no problem (well we use the running buffer with HCl-tris , glycine and SDS), but with MeOH I don´t know, may be the proteins with MeOH lose their negative charge (contributed by loading sample buffer) and it don´t run well.
When you, finish tell me about how run the gel.
good luck!

[Luthien]

But if you can change the buffer do it!

[Priya914]

I think you should re-run the gel. You cannot run the gel using transfer buffer.

[bob1]

The only difference between Towbin running buffer and transfer buffer is usually methanol which is there to stabilise the gel and strip complexed SDS from the proteins.

It is best to run the gel again, but it may run OK, though the proteins might not migrate where you expect them to.

[VJbiofication]

I did the same mistake today and I've run the gel with Transfer buffer instead of Running buffer. We don't use any SDS in the transfer buffer. But the marker bands and the Sample buffer was run perfectly as usual without any problem(by observation of the gel). And I am transferring now with transfer buffer to PVDF. I don't know how the blot is gonna look like.
Any suggestions or comments?

[VJbiofication]

VJbiofication, on 09 November 2012 - 06:46 AM, said:

I did the same mistake today and I've run the gel with Transfer buffer instead of Running buffer. We don't use any SDS in the transfer buffer. But the marker bands and the Sample buffer was run perfectly as usual without any problem(by observation of the gel). And I am transferring now with transfer buffer to PVDF. I don't know how the blot is gonna look like.
Any suggestions or comments?

luckily the gel was run fine and even the western blot turned out to be good. So I guess using transfer buffer for running is not such a big mistake.