Methods of concentrating Protein lysate - (Nov/17/2008 )
Dear all,
I just finished preparing my protein lysate from cell culture, with RIPA lysis buffer. However, the final concentration is too low to start a WB. Is there any method to concentrate my protein lysate?? Can i simply concentrate it by centrifugtion in vaccum? Is there any damaging effect to the lysate If i speedvac them?
Thank you in advance
-BBC-BBQ-
QUOTE (BBC-BBQ @ Nov 18 2008, 03:14 AM)
Dear all,
I just finished preparing my protein lysate from cell culture, with RIPA lysis buffer. However, the final concentration is too low to start a WB. Is there any method to concentrate my protein lysate?? Can i simply concentrate it by centrifugtion in vaccum? Is there any damaging effect to the lysate If i speedvac them?
Thank you in advance
I just finished preparing my protein lysate from cell culture, with RIPA lysis buffer. However, the final concentration is too low to start a WB. Is there any method to concentrate my protein lysate?? Can i simply concentrate it by centrifugtion in vaccum? Is there any damaging effect to the lysate If i speedvac them?
Thank you in advance
Tangential flow filtration will reduce the volume effectively (1L culture medium to ~20 mL in a few hours), and you can add a buffer exchange step too. Millipore, Pall and Sartorius all make TFF units. Check http://www.biocompare.com for more suggestions, or just google "Tangential flow filtration".
Ammonium sulphate precipitation will give you partial fractionation.
-swanny-
QUOTE (BBC-BBQ @ Nov 17 2008, 08:14 AM)
Dear all,
I just finished preparing my protein lysate from cell culture, with RIPA lysis buffer. However, the final concentration is too low to start a WB. Is there any method to concentrate my protein lysate?? Can i simply concentrate it by centrifugtion in vaccum? Is there any damaging effect to the lysate If i speedvac them?
Thank you in advance
I just finished preparing my protein lysate from cell culture, with RIPA lysis buffer. However, the final concentration is too low to start a WB. Is there any method to concentrate my protein lysate?? Can i simply concentrate it by centrifugtion in vaccum? Is there any damaging effect to the lysate If i speedvac them?
Thank you in advance
once I had the similar problem and my RIPA lysate wasn't concentrated enough. I used ACETON to precipitate the proteins. I added directly to my sample. then I rotated for 1 hour at 4 degrees. then centrifuged at a very high speed. then resuspended in a lower amount of RIPA.
you can modify my method, or google search for aceton precipitation.
use 20-30ug for each well. however this would depend on your application.
-Curtis-