PVDF blot storage - (Jul/16/2009 )

Hi guys,

Ran a western this week and I wont be able to come in this weekend to finish my probing. I need to store my pvdf membrane till next week but I'm not sure the best way to go about doing it. Should I...

A ) Strip membrane after my last round of ECL detection and keep the membrane in a container of wash buffer, pbs + azide, dH20?
B ) Strip membrane and dry the blot between pieces of filter paper, then rewet it on monday?

If I do B, would I reactivate the membrane with 100% methanol? Or would I just start off with washing in TBST? Just not sure what will be the best way to prevent degradattion

Any help would be mucho appreciated!

Thanks

Hi,


I suggest (A) storing the blot dry over the weekend, strip and re-probe next week when you are ready. You'll have to wet out the dry blot with 100% MeOH (then perhaps 50% MeOH in TBS-T) before putting it into 100% TBS-T. Alternativey, store blot in wash buffer over the weekend, but strip and re-probe next week.

cancergeek on Jul 16 2009, 07:40 PM said:

Thanks Jeffle.

Is there any particular reason why the blot shouldn't be stripped before storage?

There is no reason to re-wet with Methanol. The methanol step is only required so that proteins may bind to the hydrophobic membrane during transfer.


I would:
1) Store blot in TBS wash buffer over weekend
2) Strip and reprobe next week.

I do not see a problem in stripping now or later, the end result will likely be the same. Matter of preference. However, if you strip, then dry the membrane, any antibodies that were not removed from the strip will be permanently stuck to the blot (they become incorporated onto the membrane much like your protein does during transfer). This is why you should always keep your blot wet after you begin probing.