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Western Blot Forum Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


right way to assemble the sandwich

right way to assemble the sandwich - Western Blot Forum

right way to assemble the sandwich - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


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Old 09-11-2009, 02:12 AM
Pipette Filler
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Default right way to assemble the sandwich



Hmmm we have this issue in the lab with sandwich assembly.

Most of the people assemble the sandwich in the transfer buffer - literally, they fill the microwave dish with 2-3 cm of buffer and assemble within.

I noticed, that when I do it this way I get with bunch of bubbles or just more blurry blots then I would assemble on the bench, even thought I would roll the sandwich very well with a roller. I guess I pick up the buffer when I lock the cassette.

How do you guys assemble sandwich? That is a bit of a silly question, but I am qurious what is the preferred way.

Thanks!
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Old 09-11-2009, 02:45 PM
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Default Re: right way to assemble the sandwich

Ok, I just called biorad and they told me that bubbles should have round even appearance on the membrane.

With me, I have more like cracks, speckles on the membrane after I Ponceau red it.

May be someone know what it is and what can be an issue?
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Old 09-13-2009, 02:33 AM
Pipette Filler
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Default Re: right way to assemble the sandwich

Issue solved.

Apparently, the thinning pads can cause this pattern.

I changed pads today so that the sandwich is nice and thick, assembled in the buffer and got a good transfer.
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