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We prepare Jurkat -/+ Anti-CD3 antibody (10 µg/mL for 2 min) cell extracts as follows:
- Jurkat cells are cultured to the desired volume (200-400 mL) and a density of approximately 1x10^6 cells/mL, then serum-starved overnight.
- 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.
- The cells are treated with 10 µg/mL Human CD3ε Activating (OKT3) Mouse mAb (Low Endotoxin, Azide-free) (#92511; https://www.cellsignal.com/produc…
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Our CD45 (D9M8I) XP (#13917), CD45 (D4H7K) ( #72787), and CD45 (D3F8Q) (#70257) Rabbit mAbs recognize intracellular (cytoplasmic) epitopes in the human (UnitProt ID P08575) and mouse (UniProt ID P06800) CD45 proteins, respectively. The CD45 isoforms differ in their extracellular domains as a result of alternative splicing. Therefore, #13917, #70257, and #72787 are predicted to detect all of the CD45 isoforms including CD45RO and CD45RA.