CBA054 Sigma-AldrichInnoZyme™ Calpain 1/2 Activity Assay Kit

Figure 1: Human calpain 1 activity

The activity of Calpain 1, Human Erythrocytes (Cat. No. 208713) was determined as outlined in the Detailed Protocol provided above.

Figure 2: Human Calpain 2 Activity

The activities of Calpain 2, Porcine Kidney (Cat. No. 208715) and Calpain 2, Rat, Recombinant, High Purity, E. coli (Cat. No. 208718) were determined as outlined in the Detailed Protocol provided above.

Figure 3: Inhibition of Calpain 1 and 2 by Z-LLY-FMK

Calpain 1, Human Erythrocytes (Cat. No. 208713) and Calpain 2, Porcine Kidney (Cat. No. 208715) were incubated with Z-LLY-FMK (Calpain Inhibitor IV, Cat. No. 208724) at room temperature for 10 min. Following incubation the activities were determined as outlined in the Detailed Protocol provided above.

Figure 4: Calpain 1 and 2 Activity in Biological Samples

Jurkat (human lymphoblastic acute T cell leukemia), HL-60 (human myoblastic acute promyelocytic leukemia), and SK-BR-3 (human breast adenocarcinoma) cells were cultured in DMEM or RPMI medium supplemented with 10% FCS and harvested at 80-90% confluency. To prepare total cell lysates, cells were pelleted, washed, and resuspended in Lysis Buffer (CytoBuster™ Protein Extraction Reagent, Cat. No. 71009) at 200-600 µl/10⁷ cells, based on cell size. The suspension was incubated for 30-40 min on ice and centrifuged for 10 min at 15,000 x g, 4°C. The supernatant was used immediately or dispensed into aliquots and frozen at -70°C for future assay. Human spleen extract was prepared by homogenizing tissue with 170 mM NaCl, 2 mM EDTA. The protein concentration in cell lysates and tissue extracts was determined using the BCA protein assay. Calpain activity was determined as outlined in the Detailed Protocol provided above.