Polyacrylamide gel preservation

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medfreak
medfreak's picture
Polyacrylamide gel preservation

Hi all,

I've been doing my first Western Blotting these past couple of days. I'm studying some muscle proteins.
I've finish the step of runnig the gel and transferring the proteins to the n/c membrane.
However, the gel (in the sandwich) is still in the tank covered in transfer buffer.
I'm going to need to leave for about 5 ays and come back again later to finish with the western.

My question: can I leave it there inside the buffer all that time?
I'm afraid it would be ruined?!

I'd also love to know of all the ways SDS-PAGE gels can be preserved, for how long, and at what step can that happen?

PS: I've made a mistake in my pre-first attempt to do Western; I finished running the gel (electrophoresis) and I had to leave it in the buffer for 2 days (outside of the fridge!), later when I was trying to take it out to lay it on the n/c membrane in the sandwich for the next phase (transferring) it was so fragile that it tear  apart very easily!
I'm afraid of doing a similar mistake!

Chin Fen Teo
Chin Fen Teo's picture
 Hi medfreak,

 Hi medfreak,

Rule of thumb, do not save anything on the gel. Once the proteins got transferred onto a membrane, you can safely keep the membrane in TBST or PBST and start the blotting procedure once you get back to the lab.

Good luck!

medfreak
medfreak's picture
Thank you!

Thank you!

medfreak
medfreak's picture
I have another question

I have another question please.

What type of TBST buffer should I keep my membrane?
I mean is it 1X (50 mM Tris) or 10 X (500 mM Tris) or even another that is 10 mM Tris (which is the one I'm using)?

I'd highly appreciate answering this question.

samm
samm's picture
If you are staining membranes

If you are staining membranes with fluorescent Abs (for imaging on the Typhoon or Odyssey), you can actually dry out PVDF membranes between two sheets of filter paper, and store cool and dry (keeps for months). Wet it it with methanol followed by TBS before probing again with a different Ab set or reimaging.
For NC membranes, store in TBS+0.1% Na-azide for several days (my longest has been <3 weeks). NC membranes don't seem to like repeated drying/wetting cycles.

HRP labeled secondaries tend to die pretty quickly if stored wet in TBS (NO Azide!) or dried out, but you should be able to keep for a few days especially if you intend to reprobe/probe for the first time.

As pippuri said, do not bother storing the gel! Also, you can just use regular TBS (0.05M Tris) for storing the membrane - even water will do in a pinch, though I would not recommend it.

Best,
-sam

medfreak
medfreak's picture
 Thanks a lot Sam!

 Thanks a lot Sam!