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Best lysis buffer for IEF/ 2D gel?


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#1 Lyanesse

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Posted 23 March 2009 - 03:50 PM

Hello all,

I am a veteran western blotter but am about to attempt my first 2D gel. I was wondering what you all recommend as the best lysis buffer for cultured mammalian cells (neurons) for this procedure? I am first going to run my samples on an IEF 3-10 gel from Invitrogen, then fix/stain the gel, cut out the lane and then run it in a Nupage 2D Bis-Tris gel, according to Invitrogen's protocols. Ultimately I am going to be trying to detect a protein that I normally detect by western blot with a phospho-specific antibody. I am concerned that the antibody may not recognize my protein if it is not reduced, and as far as I can tell, none of the steps in the Invitrogen IEF/2D protocol mention reduction/denaturation of the sample.

Invitrogen tech support told me that a TNE-type lysis buffer with NP40 would be sufficient to prepare my sample for the IEF gel, however as I peruse the web I see that many protocols include urea and/or DTT and state that the sample must be reduced/denatured prior to IEF. ??? I am aware that the protein itself might dictate what buffer is optimal, however the problem is that I don't know much about the protein--in fact I am trying to ID it with this procedure. All I know if that it is phosphorylated and quite soluble (in TNE or TNE+TritonX).

Any suggestions or links to reliable protocols would be greatly appreciated! Thank you!!

~L

#2 neuron

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Posted 23 March 2009 - 08:40 PM

Hi,

we use lysis buffer to lyse the mammalian cell lines but in this buffer we don't include DTT, the buffer composition is-
(for 1 mL)
Phosphate buffer,pH-7(50uL)
10% deoxycholate(100uL)
10%NP 40(100uL)
10x protease inhibitor cocktail(100uL)
4M Nacl(32.5uL)
and MilliQ

after lysis we do acetone precipitation and resuspend the pellet in rehydration buffer which is Urea, thiourea and CHAPS. and reduction alkylation we do after IEF.

Hope it helps:)

#3 Lyanesse

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Posted 25 March 2009 - 01:31 PM

Thank you for the suggestions!

~L




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