Urea and Western Blot - (May/28/2008 )

I have very small tissue samples that I need to use for qRT-PCR and for Western Blots. I've been using this kit that separates out the genomic DNA and RNA and then acetone precipitation to get the protein. My main problem is what to dissolve the protein pellet in. The kit suggests 8M urea or 5% SDS. I have had bad luck using SDS to completely dissolve the whole pellet, and then there's the issue of precipitates forming when the solution is cooled. I have been trying to get information on how Urea behaves in a western and I have come up with several questions that I'm having a hard time answering.

1. Does 8M urea interfere in the gel running? Should I resuspend my protein in a small volume of 8M urea and then dilute it when I load the gel?
2. The sample buffer I use for westerns suggests heating the sample to boiling before loading the gel. I'm assuming that is because of the SDS in the sample buffer. I have read that heating urea and proteins together causes carbamylation which I'm not sure will interfere with my western. I've read that some people heat their samples to 30-37C before loading their gels, but I"m not sure what type of gel they were running or what type of sample buffer they are using. So I guess my question is, what temperature do I heat my samples to , if at all, when using proteins resuspended in 8M urea and using Laemmli sample buffer, or XT sample buffer?
3. Is there any other option for resuspending my protein pellet besides SDS and 8M urea that will not interfere with a BCA protein assay?
4. How long can an 8M urea solution bestored and at what temperatures?

That is all for now. Thanks for any help.

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Yes a little bit but not too much it's nothing compar to GnHCl for example which really alter the migration



Usually it's not necessary if you use a 2X SDS loading buffer it works OK



I don't worry about the heat treatment just 2mn at 95°C should work perfectly, I perform hundreds of Western Blot with such samples and never had any troubes in recognition ( I use mainly anti His tag mAb)



Well you can try to resuspend it in a Phosphate buffer like pBS or Tris Phosphate but for sure somme of the proteins won't be soluble

As for the Pierce BCA assay the limit of UREA is 3M so if you dilute your sample accordingly to the method (20microliters in 200microliters) they will be not too much interaction (don't forget to use the same buffer for you point Zero)

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Thank you very much for all of that info. It's nice to have it all spelled out in one place.

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hi i have a question that, i use hippocampal tissue for membrane protein detection by western blotting. but i am not able to identify all receptor molecule which are important. so If anybody have a idea about how to separate all membrane proteins. may be by changing the concentration of ingradients used. please i need advice
Thanks a lot
Sudarshan

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Please open a new subject Sudarshan it will allow other members to see it and to give you more help

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