I need to IP proteins and protein complexes from the culture supernatant. I am wondering if it is any different from IPs from cell lysates. For example, do you add the normal repertoire of inhibitors? Any experience-based advice would be appreciated.
Performing IP with cell culture media for secretory proteins could be challenging if the proteins of interest are not abundant. In this case concentrating the media using Amicon membrane of targeted cut off size could be important step otherwise you have to add more antibody which may be expensive and can cause associated problems with strong IgG heavy and light chain contaminating signals.
I frequently handle secretory proteins MMP 2 and 9 in culture and use YM10 membrane filter for concentrating the sample. Adding protease and phosphatase inhibitor cocktail in the media after collecting the sample is required to stabilize the proteins to avoid degradation by proteases. Concentering about 10-15 fold of the media works well for me.
You do not need to add conventional IP lysis buffer that usually contain combination of detergents because there is no cell for lysis. You can modify the buffer by keeping SDS only as this is required to unfold the protein of interest to expose epitopes to the added primary antibody.
Hope that helps!
The sooner the better. If you add protease inhibitors while cells are growing you may affect growth and/or protein secretion. Therefore I would just take cell culture, add cold antibody and make IP at 0-4 degrees. Eventually I would compare IP results after 2 hours and after 6-10 hours of IP.
Performing IP with cell culture media for secretory proteins could be challenging if the proteins of interest are not abundant. In this case concentrating the media using Amicon membrane of targeted cut off size could be important step otherwise you have to add more antibody which may be expensive and can cause associated problems with strong IgG heavy and light chain contaminating signals.
I frequently handle secretory proteins MMP 2 and 9 in culture and use YM10 membrane filter for concentrating the sample. Adding protease and phosphatase inhibitor cocktail in the media after collecting the sample is required to stabilize the proteins to avoid degradation by proteases. Concentering about 10-15 fold of the media works well for me.
You do not need to add conventional IP lysis buffer that usually contain combination of detergents because there is no cell for lysis. You can modify the buffer by keeping SDS only as this is required to unfold the protein of interest to expose epitopes to the added primary antibody.
Hope that helps!
I am agree with Amritlal Mandal, but I want to suggest one more thing which might you encounter during the IP. Due to use of higher concentration of Antibody for pull down you will get strong signal of heavy and light chain in blotting, to void this you can use Confirmation specific Secondary antibody, which will detect only native structure of antibody instead of binding to the denatured Light and Heavy chain of antibody you added during pull-down.
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