Protein extraction buffer compatible with 2D PAGE - (Jun/24/2010 )
I am trying to extract proteins from formalin fixed liver tissues using a 6M guainidine HCl based buffer. I did a TCA precipitation to remove the salt and then ran my IPG strip which took 2 hours longer than a run done on feline serum so I started to become concerned about salt still being an issue. Then there were no bands on my 2nd dimension (low protein conc is a possible issue here). I tried SDS PAGE on the same sample and got very smeary looking bands which pushed the broad range marker laterally. Do I have a salt issue or should I be trying a urea based tissue lysis buffer? My only concern with that is that I boil the tissue homogenate for 2 hours which I obviously can't do with urea. Would warming for a long time (ie. a day?) at a temp. below 37C be sufficient? My ultimate goal is to do 2D PAGE on these extracts. Thanks for any wisdom as I don't have much! I only started in proteomics a month ago - 3rd yr veterinary student posing as a researcher for the summer
tca precipitation will denature the proteins. you may be removing the salts but your proteins will not be very soluble (hence, the smear when you run on sds and the lack of migration in ief). residual tca (did the loading buffer turn yellow?) may also effect the way the protein runs down the lane.
you are correct that you shouldn't boil a urea solution.
instead of tca precipitation why don't you try dialysis to remove the salts.
The loading buffer did indeed turn yellow when I loaded it on the SDS gel. I tried to dialyze the sample before trying the TCA preciptation (just placed the dialysis bags into distilled water) and my samples turned into this brown chunky material within about an hour - any ideas on that one? Right now I'm trying out a urea based lysis buffer and just warming my samples at 35C for several hours, but from what I've read the guanidium based buffers do get higher yields.
try dialyzing against the buffer without guanidine.
for the gel sample, don't worry about a precipitate. it should solubilize in the loading buffer.
urea will decompose at 35C. you can stay with guanidine.
I will redo my extraction to try that dialysis next week and let you know how it goes. Thank you SO much for the advice!