Serum Preperation for 2D - (Oct/07/2005 )
I am running a 2D protocol on a serum sample. I am trying to isolate a protein with a low pI, possibly under a pH of 5. It has a low molecular weight, around 34 kDa. I have never had any luck with westerns if i remove albumin from the sample. I am also using capillary tube gels in the first dimension. If any one has knowledge of 2D, and could offer information for the best method of sample preperation, or buffers, and gel solutions for IEF, it would be appreciated. I have tried a few different methods with somewhat pour results. Thanks
Removal Albumin is the most critical factor to get a good result.
Some commerical Albumin Depletion Kits are available. You can have a try.
Up to what I know, there is NO kit which can totally remove albumin. Just try to keep it as minimal as we can.
If you would just like to isolate the protein with low pI, you can try to use IEF Fractionator.
This helps in isolating the protein at different pI in LIQUID form.
You can also load more proteins (which are within the low pI) into the same strip.
Then, you can run this sample in a narrow strip to get the highest resolution.
As the IEF Fractionator is not cheap, it ranges from (US $X,000 to $XX,000) from different companies.
You have to consider whether you will keep on this technology and the budget of your project.