5 replies to this topic

[Hummingbird2]

Hi, all - I use an older BioRad system for western blotting. The membranes I use are just a standard BioRad PVDF on a roll that I cut to size. The membranes work great all throughout the blotting process . . . unless I have to reprobe one . . .

After exposure I wash the membrane a couple time with TBS-T and then let dry out. Once dry, I re-activate with some methanol for a minute or so. Then I wash a couple times with DD H2O and put it in the 1X reprobe solution (5X solution from G Biosciences, diluted to 1X with DD H2O). The problem is, the membranes always float on top of the reprobe solution! No matter how much I poke them down or swish things around, they rise to the top again! 

I know PVDF is hydrophobic, but I never have any issues with any of the other reagents (TBS-T, antibodies, milk, etc) which are also made in DD H2O - PLUS I asked a colleague who reprobes all the time about this and she said she's never had issues with membranes refusing to submerge into reprobe solution (and she uses the same exact brand I do and dilutes it in the same exact DD H2O that I do)!

 

So what is up!? Any suggestions??

 

Cynthia

 

[mdfenko]

Does the membrane float in the water when you rinse off the methanol?

 

if not then you may be over washing after reactivating.

 

if the reprove solution contains methanol then you may want to try not rinsing after reactivating. If not then just rinse once, quickly, after reactivating.

[Hummingbird2]

Yes - it does start to float during the DD H2O washes after MeOH re-activation . . . 

 

Cynthia 

[mdfenko]

Then you’re probably over washing with water. One rapid rinse should be sufficient.

[Hummingbird2]

Okay - that's what I thought you meant, but that's kind of the opposite of what you wrote previously. I got you though! I do those washes the same way I do all my other washes and everything else is always fine (even though those reagents are also made in the same DD H2O). There must be something about washing the membranes in *just* DD H2O . . . weird. I'll just do one quick wash in DD H2O before adding the reprobe in the future! Easy fix! Thank you!

 

Cynthia

[mdfenko]

You know you only have to activate to get protein to bind to the PVDF membrane during transfer. Once transfer is complete and the membrane is blocked you don’t want anymore protein to bind to the membrane. You want the probe to bind to the bound protein of interest.

 

for a reprobe, you may only want to remove the previous probe (you may not have to depending on detection method) but not the protein bound to the membrane. You don’t want to reactivate the membrane.

 

i answered the question the way you asked it without thinking about what you were doing, sorry.

Edited by mdfenko, 07 August 2020 - 08:33 PM.