Protein extraction for 2D electrophoresis - (Aug/02/2013 )
I extracted protein using phenol followed by methanol precipitation. It has the following steps:
* On day 1, I extracted protein using extraction buffer and phenol phase precipitated with ammonium actetae in 100% methanol overnight at -20 C
On day 2, pellet was washed with ice-cold acetone/DTT and the sample was stored at -20C
My question is I have several samples to extract. Can I store just pellet at -20 C. Do I need continue the following step and stored in IEF buffer for long time? If not what is the difference between day 2 and day 3/2D separation
On day 3: Samples were cold acetone extracted via precipitation overnight at -20, the pellet was washed with 50% acetone to remove any residual contamination. Finally the pellet was dried and dissolved in IEF buffer: 8M urea, 4% Chaps, 40mM Tris-Hcl pH 8.5, 100mM DTT and 0.2% ampholyte with trace bromophenol blue (I did bradford assay here) and stored my sample for 2-3 weeks.
What I did here was, I prepared IEF buffer with all above chemicals and aliquot in eppendorf and dissolved my sample in it. Can I store sample for 2 -3 weeks or long time. If not could you please let me know the reason? Can I dissolve sample without DTT and ampholyte? or Do I need to add DTT and ampholyte before rehydrating overnight?
After extracting samples I did sample rehydration with 300microl overnight at room temperature followed by IEF and 2D electrophoresis
Could anyone check and give your valuable suggestions here.
a couple of things...
yes, you can store at -20C in the ief buffer but you should probably omit the ampholyte (normal storage is 2-8C, not sure if freezing will affect it).
bradford should not be performed in the presence of ampholytes. they bind coomassie blue. also, bromphenol blue may interfere with bradford readings and is unnecessary in ief (first dimension).
you can dissolve your proteins without the ampholyte and add it shortly before loading the gel (ampholytes may help prevent aggregation).