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ProQ Diamond Staining Issues

Posted: Mon Jul 29, 2013 9:53 am
by Calvin8
I have used ProQ Diamond Phosphoprotein stain (ProQ DPS) many times before and although I have had a few issues with background staining (swirling, white spots), most staining problems I've encountered have been overcome with increasing wash stringency and/or diluting the stain. However, I have recently encountered an unusual background staining problem with ProQ DPS stain. There is background staining all over the gel that is exactly the pattern of the bind-silane treatment (after it dried) that was applied to the glass plate. It appears that the ProQ stain bound something in the bind-silane mixture. Here is what I know:

1) The background fluorescence is present in all 9 bind-silane treated gels I am using for this experiment

2) The background fluorescence is ProQ-dependent as gels cast simultanoeus to the 9 stained gels, but NOT stained with ProQ exhibit have no signal when using the 532 laser with a 560 lp filter. Also, the background fluorescence present in ProQ-stained gels does not show up with any other laser/filter combination.

So far I have NOT moved forward with Sypro staining in the hopes that I can still get useful ProQ images. It will be interesting to find out if the Sypro stain exhibits similar background problems. I still have 3 bind silane-treated gels that I can use to help figure out what the problem is. I don't have any great ideas about the source of this problem so I'm not sure what to testThe only thing I changed in this experiment compared to the many other times that I used ProQ-DPS is that I diluted it 3-fold because several papers recommended diluting ProQ DPS 3-fold as this is more cost-effective and reduces background. I highly doubt this is the issue but...

Please let me know if you have any ProQ-DPS experience and have any suggestions or ideas on how I can use the 3 gels I have to test possible sources of background staining. Thank you for your help. :)


ProQ DPS background problem solved

Posted: Fri Aug 09, 2013 1:33 pm
by Calvin8
I have identified the source of the distinct background pattern in my ProQ DPS gels. Not surprisingly, it is the Bind-Silane. If you plan on staining Bind-Silane-attached gels with ProQ DPS, you MUST completely wipe dry the Bind-Silane after treating the glass plates with it. If you let the Bind-Silane mixture air dry, the Bind-Silane will concentrate forming a "drying pattern" on the surface of the glass plate. ProQ DPS will bind the concentrated Bind-Silane, presumably because of the similar structure/charge of Silane and phosphate. The good news is that this is NOT an issue with Sypro or Lava Purple. I started using extra Bind-Silane and letting it air dry after one of my gels came loose from the Bind-Silane-treated glass plate. This strategy is fine as long as ProQ staining is NOT in the workflow.

Now to work on strategies to salvage my gels by finding a way to selectively eliminate ProQ binding to the silane but NOT my phosphorylated proteins...