WB with cell culture medium - (Nov/17/2006 )
Hi,
I am going to analyze the cell culture medium for certain soluble proteins secreted by the cells. I would appreciate if anyone who has such experiences can give me any advice. 1) I plan to use Microcon centrifugal filter unit to concentrate the medium, but I am not sure how many fold I should concentrate it. 2) The cell culture medium contains 10% serum. I am not sure to what extent the serum proteins would interfere with the WB. 3) After concentration, do I need to determine the protein concentration with Bradford assay? Or just load as much as I can?
Any advice would be truly appreciated. Thanks in advance!
-wjchxl-
QUOTE (wjchxl @ Nov 17 2006, 11:36 PM)
Hi,
I am going to analyze the cell culture medium for certain soluble proteins secreted by the cells. I would appreciate if anyone who has such experiences can give me any advice. 1) I plan to use Microcon centrifugal filter unit to concentrate the medium, but I am not sure how many fold I should concentrate it. 2) The cell culture medium contains 10% serum. I am not sure to what extent the serum proteins would interfere with the WB. 3) After concentration, do I need to determine the protein concentration with Bradford assay? Or just load as much as I can?
Any advice would be truly appreciated. Thanks in advance!
I am going to analyze the cell culture medium for certain soluble proteins secreted by the cells. I would appreciate if anyone who has such experiences can give me any advice. 1) I plan to use Microcon centrifugal filter unit to concentrate the medium, but I am not sure how many fold I should concentrate it. 2) The cell culture medium contains 10% serum. I am not sure to what extent the serum proteins would interfere with the WB. 3) After concentration, do I need to determine the protein concentration with Bradford assay? Or just load as much as I can?
Any advice would be truly appreciated. Thanks in advance!
use a small cut-off, 1000 - 3000 Da will do
-The Bearer-
QUOTE (wjchxl @ Nov 17 2006, 04:36 PM)
2) The cell culture medium contains 10% serum. I am not sure to what extent the serum proteins would interfere with the WB. 3) After concentration, do I need to determine the protein concentration with Bradford assay? Or just load as much as I can?
2) serum proteins should not have bad effect on wb as long as your protein of interest is of a different mw.
3) you can't do a protein determination because of the serum proteins. they will overwhelm the assay.
-mdfenko-