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Can't remove Ponceau staining!


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#1 Hummingbird2

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Posted 18 May 2020 - 02:59 AM

Hi, all - First post! biggrin.png

So, 
I have always stained my PVDF WB membranes with Ponceau to get a background image, then removed the Ponceau stain by adding a bit of 100% methanol and swirling around on the membrane for a minute or so before rinsing and blocking, etc. I ran 4 WB's on Friday, added the Ponceau, and took the pic . . . but methanol won't remove the Ponceau stain! I mean, it made the red stain a little lighter, but usually 100% methanol takes the Ponceau completely off in a minute or so! I left the membranes in there for like 10 minutes - only the tiniest bit lighter!! I even dumped the methanol and tried another lab's methanol for several minutes - nothing! I have literally done hundreds of WB's and the methanol has always taken the Ponceau off *completely* and very rapidly! Any clue why it won't take the Ponceau off now!? I haven't changed anything - same membranes, same methanol, same Ponceau, same WB procedure . . . what is going on?? Thanks! . . .

Cynthia :)



#2 Hummingbird2

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Posted 18 May 2020 - 04:03 AM

Well, I washed the membranes really well with TBS-T on Friday and let them dry completely over the weekend. I just put them back in methanol and like 99% of the Ponceau came out! It took a couple minutes, but it came out! Weirdest thing ever . . . so, problem solved - but if anyone has any insight as to why this happened, I'd still be interested! Tx! 

 

Cynthia :) 



#3 bob1

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Posted 18 May 2020 - 01:33 PM

You can also wash the Ponceau off with a blocking solution - milk powder in buffer or BSA in buffer work well.






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