Basic Immunoprecipitation Question - (Aug/19/2009 )
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?
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.
After boiling, all components are separate and denatured (linearized) by the SDS in the gel. You will need to add the primary antibody to your blot.
Elution in SDS Sample Buffer with reducing agent (DTT or B-mercaptoethanol) will denature the proteins and the SDS-PAGE gel will show a linear separation based on the size of the proteins. All protein complexes will be separated, including sub-units of proteins. Ig will therefore be visible as two bands on your gel or western blot: 25 and 50 kDa for the light and heavy chains respectively. You can look up the size of your target protein in order to determine where to look on the western.
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.
Or alternatively, when you probe your western you can use protein G (or protein a) conjugated to HRP instead of a secondary antibody.
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...........