Re: Antibody-based uptake assay

Hi,

This goes way back......
I have been involved in multiple projects assessing receptor recycling 
which involves internalization, ligand dissociation and subsequent 
receptor translocation back to the cell surface.  The reference below is 
ancient but is a reasonable starting point.  The key is to use 
fluorescein conjugated antibodies because fluorescein is extremely 
sensitive to pH.  The loss of fluorescence due to internalization can be 
monitored over time under various conditions. Reanalysis of the same 
samples following fixation shows a recovery of the fluorescein 
fluorescence at the early time points.	The later time points may not 
recover total fluorescence intensity due to the surface protein-antibody 
being degraded. Fixation causes the intracellular pH to be the same as 
the buffer and the total fluorescence, both surface and internalized, 
can be measured.  We started with 20-30 identical samples dropped into a 
water bath at 37'C and acquire data for each time point; typically every 
5 to 10 minutes for the early time points extended to every 20 to 30 
minutes for the later time points.  Immediately after collecting the 
data the sample was placed on ice to stop internalization.  All samples 
were then fixed and reanalyzed.  Endosome acidification is a common 
mechanism for dissociating interacting proteins.  One can do multiple 
variations on this technique, depending on the question being asked.

good luck, TomD

Weber DA; Buck LB; Delohery TM; Agostino N; Pernis B
Class II MHC molecules are spontaneously internalized in acidic endosomes
by activated B cells.
Department of Microbiology, College of Physicians and Surgeons,
Columbia University, New York, NY 10032.
J Mol Cell Immunol 1990;4(5):255-66; discussion 266-8


Peggy Okeefe wrote:

> Hi Everyone,
>  
> I have an investigator that is interested in looking at MHC I 
> endocytosis using an antibody-based uptake assay of the cell surface 
> molecule.
> Does anyone have experience with this type of assay? If so, any 
> helpful information that you can provide would be most welcome.
>  
> Thanks in advance.
>  
> Sincerely yours,
>  
> Peggy O'Keefe
> Research Core Flow Cytometry Lab
> VASD Healthcare System
> 3350 La Jolla Village Drive
> San Diego, California 92161
> Phone: (858) 552-8585 ext. 5933
>