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.

sjouke

shubhashree:
hi
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 :'( :'(

shubhashree:
hey this the gel picture...help me....

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

sjouke

shubhashree:
hi...
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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