concentrating protein from different lysis buffers? - Can you concentrate proteins from differnt buffers and resuspend all in the same (Jun/23/2006 )
so if I have proteins over the years (whole cell lysates) that I have collected that are all in slightly different lysis buffers, and to dilute, can I somehow concentrate the protein and if you concentrate protein I guess you resuspend back in lysis buffer? So I could have them all in the same lysis buffer? I am thinking of using a multiplix system that needs to have less SDS, and I cannot maintain the correct concentration of protein if I simply dilute out the SDS.
can you concentrate them with a spin column, and then co-dialyze to equilibrate them into the same buffer?
well, there are different ways to precipitate protein:
I've tried ammoniumsulfate precipitation, chloroform-methanol-extraction and acetone precipitation and for me acetone precipitation worked best.
But you should just try
and there might always be problems getting the whole protein soluble in the new buffer again as some proteins only stay soluble with high amounts of detergents
so maybe, changing buffers could lead to decrease of protein concentration
atached are the protocols I used
of course there are also spin colums for protein buffer exchange/desalting available, for example from pierce and millipore
thanks for the protocols, I will take a look....
the problem is I kept switching buffers and now I would like to still be able to use all the proteins that I have collected, especially as some are from patient material and are precious, but I can test in my cell line stuff first, and see if I get any loss of protein during the procedure, which might actually be ok, if I can get the conc/ml down, I won't need much volume, just a highish concentration.
I just looked at the protocols, I can't believe the RLT/Qiagen one. Do you use that protocol? Do you run your protein lysates through the spin columns? Are your lysates in RLT or have you tried it with your own protein lysis buffers?? What do you use your protein for if its been extracted with RLT??
I've got my tissue extract from another department who used RLT buffer to inactivate RNAses as they wanted to do real time pcr on it.
that's the reason why I had to change buffers by acetone precipitation.
Because of the RLT buffer all protein is denatured but no prob cause I just want to do denaturating SDS-Page and Western on it...
At the protocol I start with point 2 and add 4 Volumes of Acetone to my suspension and so on...