Ponceau/MemCode and PVDF

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Ponceau/MemCode and PVDF

Postby geraldschmid » Aug 29 2003 6:31 am

There are already several "old" threads dealing with Ponceau S and PVDF membranes. Anyway, the important step is to rinse the membrane once again in methanol after blotting (1 min or so, make sure the whole membrane floats in methanol and both sides are completely covered with it).
Then rinse it again well in PBS or aqua dest (or any similar buffer). Then proceed with the staining (Ponceau S in acetic acid or TCA works equally well).
An alternative method is to use MemCode from Pierce. It's supposed to be more sensitive (not as much as they say, though) and I like the turquoise color. Anyway, they claim it does not work with PVDF membranes, but it's the same like with Ponceau: You just have to activate the membrane again before staining (with methanol) then it works just fine.
Since MemCode is also easily reversible, it's a nice alternative to Ponceau S.

A plus, Gerald
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Postby Starriedwin » Sep 05 2003 10:42 am

Hi, can I ask the reason for why the membrane need to be activated by methanol first before staining?

And can anyone suggest the recipe for the staining solution?

thanks a lot!

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Postby geraldschmid » Sep 07 2003 1:07 pm

The water and other reagents (antibodies, stain ...) can't access the surface of the PVDF membrane in case it's not activated by methanol. If you buy a PVDF menbrane (e.g. from Millipore or Amersham) just read the little manual that comes with it, it explains the properties of the membrane very well.

Anyway, even if the membrane was always kept wet, it's still important to activate it again before performing critical steps (and then of course wash away the methanol with PBS ...).

The MemCode comes with all ingredients and a manual. For Ponceau S:
1. 0.1 % (w/w) Ponceau S powder in 5 % acetic acid

or

2. 0.3 % Ponceau S in 3 % TCA (trichlor-acetic acid).

Both stain equally well. You might want to try both solutions in parallel.

Activate your membrane with mehtanol, wash it well with PBS (or water). Stain it with Ponceau solution (a few minutes) and then destain it with water (put water on the membrane, shake it a couple of times, discard water; repeat until you see the bands well and don't have much background).

With Ponceau staining it's better to scan the blot when still wet (for ducumentation) because the blot usually looks better when wet.

With Memcode you can also dry the membranes after staining because the bands are still visible well when dry.

You can even perform the staining after you already blocked the membrane and made a ECL detection.

Just strip the blot well and stain it. If you want to make another antibody reaction (... ECL), just block again and go on like usually.

A plus, Gerald
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Postby swampeee01 » Sep 07 2003 10:15 pm

Just a quick note for those of you who are, like me, still stuck using colorimetric detection rather than ECL - make sure you destain totally after the Ponceau detection. It took me a while to realise that the awful non-specific reactions I was seeing were a result of left-over Ponceau bands!
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Postby Starriedwin » Sep 07 2003 10:17 pm

Thanks, geraldschmid.

you are so helpful!

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Postby geraldschmid » Sep 08 2003 1:45 am

@starriedwin

You are welcome!

Whenever there is a clear question, I try to answer it, properly.

Beste Gruesse, Gerald
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Postby Starriedwin » Jan 26 2004 4:21 am

Hi

I have used Ponceau S to stain my membrane but I could not destain it by water, can anyone help me?

My Ponceaus receipe is 0.1% in 5% actice acid.

thank you very much

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Postby geraldschmid » Jan 26 2004 5:15 am

Hi,

usually, the destaining should work with water (it may take some time, though). But you can also try to use PBS or 5 % acetic acid.

I guess that should work well.

A plus, Gerald
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Postby Cell Man » Jan 26 2004 7:49 am

If dH20 or PBS doesn't work (or takes a long time) supplement PBS with 0.2 % Tween-20. It'll work in a fraction of the time.
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Postby r.rosati » Jan 26 2004 8:38 am

if i remember well (and i'm not pretty much sure of it), someone in the lab next to mine used to put a Whatman paper sheet together with the membrane when destaining, so that the paper would adsorb the ponceau and speedup the process.


Best luck,

-Rob
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Postby Starriedwin » Jan 26 2004 11:34 am

I don't know what's wrong with my Ponceous S solution.

It is freshly prepared today (0.1 g powder in 5% acetic acid). I stain it for around 5 minutes, then the membrane becomes all red. I can't wash out the stain on the membrane even though I finally try to use 5% acetic acid to destain.

Any suggestion?

Thank you very much

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Ponceau S

Postby adbaker » Jan 26 2004 3:00 pm

Try washing with 0.1 N NaOH, this will remove the stain completely and usually quite fast.
I use a 0.5% Ponceau stain In 1% acetic acid and I only stain for 1 to 2 minutes.

Hope this helps

Adam
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Postby Starriedwin » Jan 26 2004 9:24 pm

Hello again,

If I use nitrocellulose membrane (Bio-Rad) instead of PVDF, do I need to activate the membrane by methanol before staining with Ponceou S?

Today once I add methanol to the nitrocellulose membrane, the membrane immediately melts (becomes transparent). I don't know why and it did not happened yesterday.

Do I need to store the Ponceou S in 4C? and is it light sensitive?

Please help and thank you very much

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Postby geraldschmid » Jan 27 2004 1:55 pm

Hi, nitrocellulose does not have to be activated with methanol. That's only necessary for PVDF.

Usually, staining of nitrocellulose functions much easier but NC does have some disadvantages.

Best regards, Gerald
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Postby Starriedwin » Jan 27 2004 10:40 pm

THank you very much, geraldschmid, the information helps me a lot!

If my nitrocellulose membrane is dried, can it be blocked again and perform ECL detection of protein again?

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