AnetteM

I have tried using EndoTrap and Triton X114 but both have failed in removing endotoxins. Does anyone have a better suggestion?

/Anette

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.

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.