Can you share your protocol for preparing Jurkat -/+ Anti-CD3 antibody (10 µg/mL for 2 min) cell extracts?

We prepare Jurkat -/+ Anti-CD3 antibody (10 µg/mL for 2 min) cell extracts as follows: 

1. Jurkat cells are cultured to the desired volume (200-400 mL) and a density of approximately 1x10^6 cells/mL, then serum-starved overnight. 
2. The next day, half the Jurkat cells are collected, pelleted by centrifugation, and resuspended in 20mL serum-free RPMI 1640 media in a 50mL conical tube before treating with anti-CD3 antibody. The media volume is reduced to conserve the amount of antibody reagent required to treat the cells at a concentration of 10 µg/mL.  
3. The cells are treated with 10 µg/mL Human CD3ε Activating (OKT3) Mouse mAb (Low Endotoxin, Azide-free) (#92511; https://www.cellsignal.com/products/primary-antibodies/human-cd3e-activating-okt3-mouse-mab-low-endotoxin-azide-free/92511) for 2 minutes at room temperature in the conical tube, with gentle mixing. 
4. The treated cells are then pelleted by centrifugation, washed with 1X PBS, and lysed in the buffer of choice [1X SDS+ DTT (#7722 or #7723), CLB + PMSF (#9803), etc.]. Typically, we lyse about 2x 10^7 cells per 1 mL buffer to achieve approximately 1.0 mg/mL concentration.
5. The other half of the serum-starved Jurkat cells are collected, washed with 1X PBS, and lysed as the untreated pair. Typically, we lyse about 2x 10^7 cells per 1 mL buffer to achieve approximately 1 mg/mL concentration. 
6. The treated and untreated lysates are sonicated on ice using a probe tip sonicator (3 times for 15 seconds each). 
7. If prepared in SDS +DTT lysis buffer, the lysate is boiled at 95 - 100C for 5 minutes before use. 
8. Lysates may be used immediately or frozen at -80C for future use. 

Last updated: September 12, 2024