Basic Immunoprecipitation Question
Posted 19 August 2009 - 12:52 PM
So I'm doing immunoprecipitation to try and isolate a specific protein from a cell lysate in order to detect it through Western Blotting. My question is, when I have gone through the whole process and added Protein A sepharose beads to my Ab/protein complex and presumably obtained a Protein A/Immunocomplex, and I then boil the sample to denature the beads and have them release the immunocomplex, am I left with the Ab/Ag complex, or a mixture of Ab and Ag. Thus, when I run a Western am I looking for a molecular weight of the two combined or my protein alone. If the whole complex travels together, then does that mean that I wouldn't need to apply my primary antibody again, and that I could just add my secondary antibody and proceed to staining?
Posted 19 August 2009 - 02:42 PM
After boiling and denaturation, you are looking at a combination of both Ag and Ab. You need to add a primary antibody that detects your protein of interest. It is better to use a different detection antibody (ie, not the IP antibody) to detect this protein. If your WB detection Ab is raised in the same species as your IP antibody, then your secondary Ab will detect both Abs.
Posted 19 August 2009 - 05:04 PM
Posted 09 September 2009 - 05:48 AM
If your target protein is of similar size to Ig H or L chains you should consider crosslinking your primary antibody to Dynabeads Protein G.
Posted 09 September 2009 - 05:00 PM
Protein G (and protein A) only recognize the native conformation of the antibody. They can bind the primary antibody used to probe the blot but not the denatured antibody from the IP.
This is a significantly cheaper option...........