Author Topic: Basic Protocols for 2- D Electrophoresis  (Read 9333 times)

Sjouke Hoving

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Basic Protocols for 2- D Electrophoresis
« on: September 14, 2010, 04:51:51 AM »
Dear all,

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

sjouke

shubhashree

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Re: Basic Protocols for 2- D Electrophoresis
« Reply #1 on: December 17, 2010, 06:38:36 AM »
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

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Re: Basic Protocols for 2- D Electrophoresis
« Reply #2 on: December 17, 2010, 06:57:20 AM »
hey this the gel picture...help me....

Sjouke Hoving

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Re: Basic Protocols for 2- D Electrophoresis
« Reply #3 on: December 17, 2010, 11:53:56 AM »
Read the protocols... it doesn't mention the precipitation step with TCA. Why are you adding this step anyway?

sjouke

shubhashree

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Re: Basic Protocols for 2- D Electrophoresis
« Reply #4 on: December 17, 2010, 11:06:52 PM »
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.

Sjouke Hoving

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Re: Basic Protocols for 2- D Electrophoresis
« Reply #5 on: December 20, 2010, 12:47:50 AM »
Still don't understand... you can wash the E. coli pellet extensively with ice cold water before lysing.

sjouke

Andrzej C.

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Re: Basic Protocols for 2- D Electrophoresis
« Reply #6 on: June 15, 2011, 08:33:54 AM »
Hi All :)

I'm PhD student. I'm going to separate by 2DE renal cortex and medulla tissue and I'm not sure which technique should I use to remove salt especially from medulla. I would like to avoid usage of ProteoMiner, but standard acetone precipitation is not enough. I thought about one solution but I'm not sure if it won't be mistake:
after homogenisation of renal tissue with lysis buffer I would add 1ml of d.water, next, I would use MILLIPORE centrifuge tube with cut-of point 3kDa to remove water and (I hope) salt - this step I would repeat two or three times, next acetone precipitation and later vertex with lysis buffer. What do you think about these steps?

Kind regards,
Andrzej

P.S.
Sorry for my bad English...

Christelle

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Re: Basic Protocols for 2- D Electrophoresis
« Reply #7 on: December 02, 2011, 02:46:40 PM »
Thank you Sjouke!  :)

Christelle



Dear all,

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

sjouke

Sole

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Re: Basic Protocols for 2- D Electrophoresis
« Reply #8 on: November 13, 2013, 12:18:22 PM »
Thank you! Do you have a protocol to perform a 2D gel phosphoprotein western blotting?