IP with Monoclonal Antibodies - (Jul/02/2008 )
Hi everyone,
I'm at my wits end with this one and need some serious help before I go mental!
I've generated some (hopefully) novel antibodies against a particular cell type i'm interested in, the only thing is, I don't know what my antigen is. I'm assuming its cell surface as the antibodies work well for FACS. I've tested them with immunofluorescence on cryosections and fixed cells and they work beautifully...the only problem is, I can't figure out what they're staining.
For the last few months I've been trying to IP and silver stain the products to get them off for mass spec analysis, but I can't get a product. I've tried various lysis buffers and preparations (RIPA, TX-100, TX-114, denaturing, non-denaturing, phosphate and tris-based buffers) but can't seem to get a band visible by silver stain or western. I think being a monoclonal, there might be problems with affinity so I was wondering if anybody has any good protocols for hard-to-use-with-IP antibodies. 
Ben
I'm at my wits end with this one and need some serious help before I go mental!
I've generated some (hopefully) novel antibodies against a particular cell type i'm interested in, the only thing is, I don't know what my antigen is. I'm assuming its cell surface as the antibodies work well for FACS. I've tested them with immunofluorescence on cryosections and fixed cells and they work beautifully...the only problem is, I can't figure out what they're staining.
For the last few months I've been trying to IP and silver stain the products to get them off for mass spec analysis, but I can't get a product. I've tried various lysis buffers and preparations (RIPA, TX-100, TX-114, denaturing, non-denaturing, phosphate and tris-based buffers) but can't seem to get a band visible by silver stain or western. I think being a monoclonal, there might be problems with affinity so I was wondering if anybody has any good protocols for hard-to-use-with-IP antibodies.
Ben
Since you think it stains cell surface, your protein is more likely to be a membrane protein. So, you need to do everything accordingly.
1. Sonicate
2. Do not boil
3. Use low % gel, as well as gradient gel to make sure it is not too small, too large
4. Do a slot blot to make sure you are at least pooling it down.
5. Use the cells that best express this antibody-target.
6. Don't waste your career, do it for a month and then leave it alone!