Endocytosis Assay Problem - (Aug/13/2004 )

I have tagged a protein on the amino terminal end with a 3XFLAG tag. I have detected the protein using a confocal microscope by tagging the protein using an anti-FLAG primary followed by an Alexa-Fluor 488 anti-mouse antibody. I have done this 2 ways...by adding the antibody to the live cells over night and by permeablizing the cells then adding the primary followed by secondary. I know through these two assays that the protein is expressed on the surface.

I now wanted to see if different mutations in the protein alter the endocytosis rate. I followed a the following protocol.

1. Wash with PBS
2. Wash with 4C PBS
3. Incubate the cells with Primary (Anti-FLAG) for 1 hr @ 4C
4. Wash with 4C PBS 3x
5. Wash with 37C Media
6. Add 37C media and Incubate @ 37C for different time intervals
7. Wash with 4C PBS 2x
8. Fix Cells with Paraformaldehyde and Triton X-100
9 Continue with normal secondary labeling etc.

For some reason the Primary antibody seems to not bind @ 4C. I dont get any fluorescence when I look under the microscope. I did a control plate in which I labeled at room temp which was positive.

I was wondering if anyone had a protocol for an endocytosis assay that does not require a 4C binding period. I understand that most protocols require the 4C incubation to inhibit endocytosis and to allow start periods for the time points. Also does anyone think that it could be the antibody for some reason...sigma has different monoclonal antibodies to the FLAG epitope. Thanks so much for the help.

Have you tought about protein biotinylation??
You can biotinylate you surface protein using NHS-biotin after stimulation or not as a control, and then immunoprecipitate with streptavidin coupled to sepharose or agarose... run that on a SDS-PAGE and blot for your protein. You'll see if stimulation (or wathever you do!!) activates endocytosis as less surface protein will be found...

We used this technique very often and it works great...

Simon