protein precipitation for mouse liver - (Feb/17/2006 )

Hi,

I am trying to do 2D-PAGE analysis on mouse liver. Although I loaded 100ug on the gel I had very few spots. The protein concentration was calculated prior to TCA/acetone protein precipitation. I wondered if I was loosing protein during protein precipitation so ran what should be 15ug on a 1D gel (allowing for 10% loss of protein). The bands on the 1D gel do look very faint. Has anyone used a different precipitation method on liver with better results/less loss ? Any advice would be really appreciated!

Thanks
Anthea

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Hello,
i have similar problem, too...

i use cho cell (ChoA) as material
lysate , and get thiol proteins from sepharose 4B column
then concentrate it by TCA precipitation
and resuspend the pellet with 2D sample buffer(urea within).
but i can't disovle all the pellets
therefore i have to quantified the solublized proteins to know how much soluition will be loaded

because of the 2D sample buffer(?),
the background was too high that Bradford(Bio-Rad protein assay) was not allow to exam the protein quantity

so i want to ask...

1. (to the original poster)were the proteins after TCA/acetone precipitation resuspended and disolved well~~?
2.can any possibility that the proteins be quantified after precipitation and dissolved in 2Dsample buffer~~?
3.and some other alternitives for TCA precipitation(one specific to thiol protiens will be great!!) and quantification methods.

thank you so so so much~~~

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