I try very hard to detect CD3ζ chain phosphorylation, but until now, I still can‘t get anything. Here is my TCR stimulation protocol:

1. I resuspend the thymocytes at 1x 107 cells/mL in RPMI media. And then pre-chill thymocytes on ice for 2h prior to use.

2. Add anti-CD3 antibodies to the cells (1 μg of each antibody per 100 μL of thymocytes(about 8X10^6, the volume is 100ul)). Mix and incubate on ice for 15 minutes. 

Add 1 mL of cold medium to per 100 μL of cell suspension.

Centrifuge at 4°C, 6000rpm for 5 minutes.

Keeping the samples cold, remove the supernatant.

Gently resuspend the cell pellet.

6. Resuspend the cells in a volume of 100ul

Add goat anti-mouse Ig antibody to the cells (1 μg per 100 μL ). Mix and incubate on ice for 15 minutes.

Stimulate the cells by transferring the tubes to a 37°C water bath and incubating for an appropriate length of time.

after that, directly add the lysis buffer which contains phosphatase inhibitors to lysis the sample and keep the sample on ice about 30min. Then centrifuge and add loading buffer to boil the sample then do the western blotting.

but the problem is i can't promote the CD3ζ chain phosphorylation(like the attached shows). So do anyone have any suggestions about promoting CD3ζ chain phosphorylation?