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Ponceau staining after primary and secondary?


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#1 mhkung

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Posted 13 May 2009 - 12:29 PM

Hi all,

So I am doing a blot for the NRF2 target gamma-GCS (73KDa). After doing ECL with both the Pierce Pico and Femto kits, nothing came up! I suspect a bad secondary Ab--it's been a while since I've used this one.

Anyway, on a hunch, I decided to use Ponceau S to stain my nitrocellulose membrane. I see faint smears of red in the lanes (good blocking), and two clear bands--one at ~42 (b-actin?), and one at ~73kda (gamma-GCS???). Furthermore, in my treated cells, where I would expect to see induction of g-GCS, the ponceau stains darker! The 42kda band is of equal intensity throughout. In other words, the ponceau-stained band at ~73KDa perfectly matches the size and pattern I would expect to see if my ECL had worked.

Anyway, has anyone else ever tried this? Could I be detecting my primary, and maybe my secondary ab bound to my protein of interest? I'm currently stripping now--will restain to see if the blot looks the same. Not that I would forgo doing proper ECL detection, but oftentimes we get blank films and don't really know what went wrong. This could be a way to help figure it out.

#2 dtimm

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Posted 13 May 2009 - 02:03 PM

Ponceau is not specific enough to determine changes in expression. You are probably staining other proteins the same size, along with your target protein.

#3 mhkung

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Posted 14 May 2009 - 09:30 AM

Ponceau is not specific enough to determine changes in expression. You are probably staining other proteins the same size, along with your target protein.

If that is true, then how does ponceau staining help to determine if a gel was equally loaded?
More protein=darker stain, correct? So if there is a change in expression of a protein, that would also correlate with a change in staining intensity.
I am also skeptical about these bands, but I don't think your statement is true.

#4 Aris

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Posted 14 May 2009 - 02:50 PM

Ponceau is not specific enough to determine changes in expression. You are probably staining other proteins the same size, along with your target protein.



so what would you do? do you have another method? I use Ponceau S too and sometimes Coomasie

#5 bob1

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Posted 14 May 2009 - 04:32 PM

You should ponceau before blocking, as the presence of block on the membrane will increase background.

To determine changes in protein expression you need to do western blot - it is specific for your protein, which ponceau is not. You can use ponceau to judge overall protein expression, but not for specific proteins. Most people just use ponceau to determine whether they have evenly loaded their protein.

If you want to test this info - run a gel, coomassie stain, cut out a band and then run it through a mass spec. Any bands from between 20-90 or so kDa will most likely have several proteins present.

#6 Aris

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Posted 14 May 2009 - 09:07 PM

You should ponceau before blocking, as the presence of block on the membrane will increase background.

To determine changes in protein expression you need to do western blot - it is specific for your protein, which ponceau is not. You can use ponceau to judge overall protein expression, but not for specific proteins. Most people just use ponceau to determine whether they have evenly loaded their protein.

If you want to test this info - run a gel, coomassie stain, cut out a band and then run it through a mass spec. Any bands from between 20-90 or so kDa will most likely have several proteins present.



I agree, Ponceau is used mainly to determine the transfer and mostly the loading and not proteins of interest. It could give you and overall idea on whether you have protein or not at the MW of interest but on the blot are a couple of hundrend of proteins that have similar MWs. So i wouldnt go and do a quantification just based on the stain. If it was so we wouldtn be doing WB at all.
Now about tha black fims, sometimes tha stripping doesnt go well. Also you have to check the I and II Abs as well as the dilutions. Some II Abs are very sensitive, for example the anti-goat II Abs, so the have to be diluted up to sometimes 1:20000.




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