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Western Blotting


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#1 moerae

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Posted 20 February 2012 - 05:37 PM

Hi all,

I just did my first electro-transfer of my proteins from SDS-PAGE to PDVF membrane, the pre-stained ladder transferred really well so I didn't do a Ponceau-S stain. Out of curiosity I stuck in the SDS-PAGE to stain in coomassie and found that the gel still had a lot of protein left over. So the transfer did not work... and I'm not sure whether to carry on or not... and whether I can do a Ponceau-S stain after blocking. I also was wondering if PVDF membrane needs to be used with CAPs transfer buffer or can I use the Tris-glycine transfer buffer (which I used for this one)? I'm trying to figure out what has gone wrong with this first blot.

#2 casandra

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Posted 20 February 2012 - 07:27 PM

Hi all,

I just did my first electro-transfer of my proteins from SDS-PAGE to PDVF membrane, the pre-stained ladder transferred really well so I didn't do a Ponceau-S stain. Out of curiosity I stuck in the SDS-PAGE to stain in coomassie and found that the gel still had a lot of protein left over. So the transfer did not work... and I'm not sure whether to carry on or not... and whether I can do a Ponceau-S stain after blocking. I also was wondering if PVDF membrane needs to be used with CAPs transfer buffer or can I use the Tris-glycine transfer buffer (which I used for this one)? I'm trying to figure out what has gone wrong with this first blot.

PVDF with Tris-Glycine buffer shldn't be problem but did you prewet it in alcohol? It would be better If you can also post your transfer conditions eg how long you transferred, the voltage so the other members here can help you better... also the proteins in your gel post-transfer...are they the high MW ones?

Edited by casandra, 20 February 2012 - 07:30 PM.

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#3 mdfenko

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Posted 21 February 2012 - 01:02 PM

there's almost always protein left behind in the gel (you probably also saw some ladder in the stained gel despite seeing it on the membrane).

once blocked you can't stain with ponceau. but you should continue to process the blot.

you can adjust transfer time or current density to enhance transfer, if necessary, but you will probably always see some protein left behind, especially if it is high molecular weight.
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#4 moerae

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Posted 21 February 2012 - 04:49 PM

Hi,

I carried on with the blotting and there are very faint bands, I'm going to expose the membrane for longer. However, I still need to address the transfer problem. Sorry if I didn't provide all the information before but here it is now.

I ran my samples in a 12% SDS-PAGE gel. Made the Tris-glycine transfer buffer fresh and soaked my PVDF membrane (bought in 2007, I have no idea if the age of the membrane is a factor), in methanol for 5 mins and rinsed with water before letting it soak in my transfer buffer. Soaked all my filter paper/sponges in transfer buffer as well. Then sent the whole thing up. Ran the transfer at 100V for 1 hour and carried on from there.

I stained my SDS-PAGE gel with coomassie, which I've attached. The ladder has transferred really well so that's not there anymore.

Attached Thumbnails

  • coomassie WT N-his dil after transfer 21-2-12004.jpg


#5 science noob

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Posted 21 February 2012 - 06:51 PM

Hi,

I carried on with the blotting and there are very faint bands, I'm going to expose the membrane for longer. However, I still need to address the transfer problem. Sorry if I didn't provide all the information before but here it is now.

I ran my samples in a 12% SDS-PAGE gel. Made the Tris-glycine transfer buffer fresh and soaked my PVDF membrane (bought in 2007, I have no idea if the age of the membrane is a factor), in methanol for 5 mins and rinsed with water before letting it soak in my transfer buffer. Soaked all my filter paper/sponges in transfer buffer as well. Then sent the whole thing up. Ran the transfer at 100V for 1 hour and carried on from there.

I stained my SDS-PAGE gel with coomassie, which I've attached. The ladder has transferred really well so that's not there anymore.


Comment 1: Age of PVDF shouldn't matter (check the packaging for any 'use-by dates') if kept appropriately in the inactivated form. I'm pretty sure PVDF is activated in absolute methanol for not more than 15s, then washed in water (5 min) followed by transfer buffer (5 min) according to the manual (I use Millipore membranes). Not sure what over-activating it will do to your membrane.


Question: What is the size of your protein of interest? Have you ran a control (actin/tubulin/GAPDH)?

Question: What is the composition of your transfer buffer? And did you run it at 4oC?

#6 moerae

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Posted 22 February 2012 - 12:50 PM

I'll do that with the PVDF membrane. My protein size is 50 kDa, it's tagged with His, so I'm using anti-his antibody. Transfer buffer is made up of 25 mM Tris, 192 mM glycine and 20% methanol. The transfer was run at room-temp with lots of ice packed around it for 1 hour. I'm planning on doing an overnight transfer today and see if that improves it.

#7 mdfenko

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Posted 22 February 2012 - 12:54 PM

here are a couple of handbooks for you. they will help you to revise your protocol with explanations:

ge western blotting handbook

and

Attached Files


Edited by mdfenko, 22 February 2012 - 12:55 PM.

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#8 moerae

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Posted 22 February 2012 - 04:28 PM

Thank you. I'll take a look at these.

#9 moerae

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Posted 23 February 2012 - 07:36 PM

Ok, so I've done the transfer overnight at 30V and the transfer is better. The coomassie stain at the end showed less protein but there's still protein left over. I've also done a Ponceau-S stain of the PVDF after transfer and it's a bit smeary and horrible, but shows that there is transfer. However, the transfer still isn't great. I'm thinking of adding SDS to the transfer buffer (as the manuals said this will be better for transfer with PVDF membranes), but I don't want to do overnight transfers just to achieve this (it means almost 4 days worth of work just for what a Western to be carried out). If I do transfers at 100V and can I do it for more than 1 hour?

Also I could try CAPs buffer, but I can't figure out what the benefits of this buffer will be to me compared to the Towbins buffer with SDS when my protein is around 50KDa and PI is around 5.5 (theoretically speaking, Towbins with SDS should do the trick).

So my questions are.... Towbins with SDS (0.05%) should do the trick? Give CAPs a try instead of Towbins with SDS? Just use nitrocellulose membrane instead?

From what I can tell I don't think it's the membrane that's the issue... but I'm new at this and I'm pretty much up for trying everything. I just want to cut out trying stupid things when I can cut straight to the issue.

(Note: The coomassie gel is back-to-front... the ladder actually transferred over fine).

Attached Thumbnails

  • Coomassie tranfsfer test after transfer 24-2-12010.jpg
  • Ponceau-S stain 24-2-12008.jpg

Edited by moerae, 23 February 2012 - 07:37 PM.


#10 mdfenko

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Posted 24 February 2012 - 12:36 PM

the sds should do the trick. it's usually used for high molecular weight proteins (higher than yours) but the 12% gel is very restrictive so the sds should help.

you may be able to go more than an hour at 100V but you'll have to determine that for your system. keep in mind that the sds will make the protein move faster.

what size pore is the membrane? if it is 0.22um or smaller then you should be able to catch the protein. if it is 0.45um then you may not want to go longer. you can put a second membrane behind the first to see if any protein blows through.

you shouldn't need to switch to a nitrocellulose membrane.
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#11 moerae

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Posted 04 March 2012 - 06:49 PM

SDS has solved the problem! Thank you for the help!




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