General Category > 2-D Electrophoresis

Basic Protocols for 2- D Electrophoresis

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Sjouke Hoving:
Dear all,

I have attached two protocols that describe in detail how to prepare a sample and to run the gels.


i have started working on 2D. In my first gel I got a very unusal pattern. there was no distinct spots nd i cd only see smear on the basic end. I m using E.coli culture where the cell pellet is lysed using the lysis buffer(Urea-8M,CHAPS-2%,DTT-65mM,Bromophenol blue0-0.002%). then i perform the TCA precipitation and the protein pellet is resolublised in Rehydration buffer(supplied with ReadyPrep 2D starterkit,BIORAD). I had used a 7cm strip(3-10 pH) and loaded 125µl(169µg protein) sample. the IEF conditions are
step1 250V / 20 min / linear
step2 4000V /2h / linear
step3 4000V / 10,000Vh / rapid (as specified by starterkit manual)

the second dimension was run on 12% SDS PAGE. please let me know where i m going wrong and why m getting such pattern.please suggest some modifications.

reply please :'( :'(

hey this the gel me....

Sjouke Hoving:
Read the protocols... it doesn't mention the precipitation step with TCA. Why are you adding this step anyway?


my E.coli cultures are from High cell density bioreactors where complex media is used. so in order to avoid salts and other contaminants i have incorporated TCA precipitation step.


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