Storing PVDF membrane Help! - (Nov/11/2005 )
Hi,
I started a 4 day western blot earlier this week probing for phosphorylated and total forms of a protein as well as B-Actin (same band size unfortunately so couldn't run it with anything else) as a loading control. I realized on day two that we were running low on chemiluminescence reagents and the order was supposed to come in today (final day of the western) but it seems as though the carrier doesn't deliver on the holliday :x
Can a probed PVDF (already incubated with the primary and secondary) be left @ 4C in PBS or some buffer over the weekend and be salvaged? Ideally, although perhaps not realistically, I'd like to just start the chemiluminecse on Monday but if necessary could I strip/reprobe a membrane that has been sitting for so long? Will I run into deterioration problems? Has anyone tried this before?
-CW
I have seen people leave the membrane in block over the weekend, but not this... I am also worried that it is both primary and secondary... 
I would say keep the volume low and cover well to prevent drying out... if your volume is low enough and is stored at 4C maybe the concentration will allow primary and secondary to remain associated with the protien??? Has to be at 4C so the enzyme doesnt stop working...
I think small volume and go for it monday with chemilum.. the worst that happens is you have to strip and reprobe...
Dont add the secondary and primary...just add some ttbs (5 ml) to the pvdf in a kapak puch...seal it an put at 4C. Even if you already have then just pour some ttbs...i am not sure though if u can get away with just chemi-iing the membrane. I normally dont do this as I look for phospho proteins...they might not give u the exact signal....however. I do it say for total and B-actin.
I normaly save an already probed membrane...and if i need to detect additinal proteins..then I just strip it, block it and add so on...
good luck
Thank you very much for the responses!
Just curious Pria, whats the longest that you have stored a pvdf membrane with the intention of reprobing at a later date?
Just curious Pria, whats the longest that you have stored a pvdf membrane with the intention of reprobing at a later date?
If its B-actin...a week, but other protein especially AKT, ERK then 2-3 days not more. I have heard that one of my seniors used to probe it after months...but thats just heresay :-)