Internalization assay for antibody internalization? - (Nov/14/2012 )

I am trying to set up an assay to study the internalization of an antibody into HEK cells. The antibody binds to a surface exposed receptor and is proposed to be internalized upon binding.
I have so far tried to incubate the cells with biotinylated antibody (which clearly binds to the cells) and thereafter stripp the cells from surface bound antibodies followed by cell lysis. I have however not been able to get a positive result.
I am unsure of if my assay set up is ok regarding amount of cells, lyzis buffer, stripping buffer etc.
I do not have any positive control either so it is difficult for me to detect where the problem is.

Does anyone have a good protocol of an internalization assay?
Or does anyone have any hints or tips to give me?

Thankful for all help I can get
/Anette

I assume you do western blot after lysis, correct? Choosing the right lysis buffer is very important in such experiments. My question is why don't you run immunofluorescence experiment with your cells? this way you can view where the antibody binds to. What is your protocol, I'll have a look at it.

Have you thought about using flow cytometry?

AnetteM on Wed Nov 14 12:53:53 2012 said:

I forgott to say that I do not have acces to a flow cytometer so I feel more or less obliged to use some kind of ELISA based assay.

Here is the protocol I use (briefly)

Maybe it is easier for someone to give me comments when you see that.

Internalization assay with biotinylated antibody and cell lysis.
Buffers
Buffer A: 0.1M PBS pH 7.4, 1.0% BSA
Stipping buffer. 50 mM TCEP, 150 mM NaCl, 1.0 mM EDTA, 0.2% BSA, 20 mM Tris, pH 8.6 made fresh)
Lyzis buffer: 20 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, protease inhibitor cocktail
ELISA wash buffer: PBS-Tween tabletter dissolved in MQ H₂O, {PBS, 0.05% Tween), Medicago #09-9410-100)

Proceedure:
- Start internalization by resuspending the cells at a density of 1 *10⁶ cells/ml (100µl/aliquot) in 37 °C medium supplemented with antibody at a concentration of 15µg/ml.
- Incubate cells at 37°C for various times. Include a negative control kept on ice the entire time.
- Stop internalization after 5, 10, 20 and 30 min by adding ice-cold buffer A.
-Wash cells twice in ice-cold buffer A.
- Stripp the cells from non-internalized biotin by resuspending in 0.5 ml cold stripping solution (50 mM TCEP, 150 mM NaCl, 1.0 mM EDTA, 0.2% BSA, 20 mM Tris, pH 8.6) and incubated on ice 15 min, with gentle shaking.
- Spinn down cells and resuspend in 0.5 ml fresh stripping solution and incubated an additional 15 min on ice.
- Wash cells twice with 0.5 ml cold buffer A
- Lyze cells by resuspending in 200µl lysis buffer (20 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, protease inhibitor coctail) and incubate 20 min on ice.
- Applyed cell lysate (100µl/well) on ELISA plates coated with neutravidin.
- Incubated 1 hour at RT.
- Wash with ELISA wash buffer and incubate 1 h at RT with AP conjugated secondary antibody.
- Wash with ELISA wash buffer and add detect by adding substrate and measure absorbance at 405 nm.