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Electrophoresis


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10 replies to this topic

#1 cariboeck

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Posted 13 January 2005 - 05:28 AM

Hi! I have used transfer buffer (Tris/SDS/glycine/10 % methanol), but after transference I have no seen staing with Pounceau in my nitrocellulosis membrane, even I see staing with the Ab. Is there any methodology to solve this trouble?
Thanks
Cari Boeck

#2 Sprag

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Posted 14 January 2005 - 11:44 AM

Maybe you are not adding enough protein on your gel?!.. Ponceau is not as sensitive as WB!!!

Also check if you really use nitrocellulose and not PVDF!!..

I've had problems with ponceau stainings of PVDF and instead of the standard Sigma (0.1% ponceau, 5% acetic acid), i made my own ponceau as 0.2% ponceau in 3% TCA, and that worked.

#3 littlecell

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Posted 14 January 2005 - 01:15 PM

i am afraid your PONCEAUS has been expired and need a fresh one.
in addition, don't use SDS in your transfer buffer again. it's not useful for most protein transfer.

Edited by littlecell, 14 January 2005 - 01:16 PM.


#4 fred_33

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Posted 01 February 2005 - 04:05 AM

I DO NOT USE SDS (important) and don't use methanol (optionnal) for my transfert solution, and everything is ok.

running :

glycine 1,92M
SDS 1%
tris base 250mM
for one liter, it is 144.13g 10g and 30,28g respectively

transfert :
glycine 1,92M
tris base 250mM
for one liter, it is 144.13g and 30,28g respectively

If you want, you can add methanol :
10X transfert solution 100 ml
methanol 200ml
ddh2O qsp 1 liter.

For ponceau solution :
0,2% ponceau red
1%acetic acetique

Good luck

#5 Terente Sangre

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Posted 03 March 2005 - 10:03 AM

Yep, I had the exact same problem! I was trying to stain the PVDFs with Ponceau, as someone else on the floor was doing, and to no avail. I was told that you take the membranes from the transfer buffer and immediately immerse them in Ponceau. It just wasnt working.
Anyway, I started to play with it and I discovered that if you take the membrane and wash it with 100% methanol for a few seconds and after that you immerse it in Ponceau, and then you take it back to methanol, the bands begin to stain right in front of your eyes. (Its like developing photo-film). Im glad to see that other people are doing the same.
What I don't understand is why does it need re-hydration, as at no step during the transfer protocol is the membrane allowed to dry out? Just curious...

#6 fred_33

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Posted 04 March 2005 - 12:49 AM

hi
maybe SDS and ponceau Red are uncomatible...
By wahshing your membrane in 100% methanol, you get the majority of SDS in methanol (let say 90%). Then, when staining in ponceau red, the last 10% of SDS gets slowely in solution, that's why bands are appearing like photo devlopment. In fact, i suppose it beacause i do not use SDS and when i stain in ponceau, bands appears immediately (less than 2seconds)!
Any idea on that ?

Fred

Edited by fred_33, 04 March 2005 - 12:52 AM.


#7 minylim

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Posted 14 September 2009 - 03:39 PM

I'm confused...nitrocellulose doesn't need MeOH activation, right Fred?

I'd doubt whether I apply current right direction and then will suspect the sample. SDS is not a critical factor on my experience still you may want to skip it in transfer buffer. Good luck!

#8 rozer henry

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Posted 16 October 2009 - 09:11 AM

I'm confused...nitrocellulose doesn't need MeOH activation, right Fred?

I'd doubt whether I apply current right direction and then will suspect the sample. SDS is not a critical factor on my experience still you may want to skip it in transfer buffer. Good luck!


nitro cellulose is entirely diffrent then methyl hydroxide... the current direction is ok... it will take some time... have patience... Rozer Online pharmacy

Edited by rozer henry, 16 October 2009 - 09:16 AM.


#9 Helios

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Posted 01 November 2009 - 01:37 AM

ya nitrocellulose doesnt require methanol....infact the membrane can be spoiled in 100% methanol......just wash your membrane after transfer with PBS and then immerse in ponceau....i always do tha for my membranes....also you can load a prestained marker in the gel which can be transferred to the membrane....that ways you can be absolutely sure that your transfer has worked...

#10 jaggsen

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Posted 16 March 2010 - 09:32 PM

Gel electropheresis allows us to study DNA by separating nucleotide strands of different lengths. Therefore, we can use different enzymes to cut DNA and observe the resulting bands that show up in electrophoresis. This can tell us, for example, where certain types of sequences happen on the genome.

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#11 50dolars

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Posted 30 September 2010 - 03:57 AM

You check the level of protein

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