CBA020 Sigma-AldrichK-LISA™ Checkpoint Activity Kit

Table 1: Preparation of Reagents

Prepare all reagents immediately prior to use. The above table provides reagent preparation instructions to obtain the Volume of Working Solutions (WS) required for one 8-well strip.

Table 2: Order of Reagent Addition Protocol A

*For inhibitor screening assay; if no inhibitor is to be included, up to 20 µl Chk Sample can be added or inhibitor can be substituted with 10 µl dH₂O.

Table 3: Order of Reagent Addition Protocol B

Figure 1: Activity of Purified Chk1

The activity of His•Tag^®, Human, Recombinant Chk1 (Cat. No. 220479) (17-2200 pg) was determined using protocol A as indicated in the Detailed Protocol section. Assay range: 34 pg-1100 pg (740 Units/mg).

Figure 2: Staurosporine Inhibition of Chk1

The activity of His•Tag^®, Human, Recombinant (Cat. No. 220479) (1.1 ng) was determined as in Figure 1 in the presence of increasing Staurosporine concentration.

Figure 3: Activity of Chk1 Immunoprecipitated From HeLa Cell Lysate

Cell lysate was prepared using 0.5 ml PhosphoSafe™ Lysis Buffer (Cat. No. 71296) for each 10 cm cell culture dish, according to the standard protocol. Cell lysates were either used immediately or stored at -70°C until use. Equal amounts of total protein (500 µg) were immunoprecipitated using the indicated antibody and activity was determined according to Detailed Protocol B.

Figure 4: Assay Characteristics and Examples

Inhibition of immunoprecipitate-associated Chk1 kinase activity from HeLa cell extracts and Chk1, His•Tag^®, Human, Recombinant (1.1 ng) using the Chk1-specific inhibitor, SB218078, and staurosporine, respectively. Immunoprecipitation of Chk1 was performed as in Figure 3. Kinase assays were performed either in the presence or absence of 10 nM staurosporine for purified Chk1 as in Figure 2 or 100 nM of SB218078 (Cat. No. 559402) for immunoprecipitated Chk1.

Figure 5: Activity of Purified Chk2

The activity of Human, Recombinant Chk2 (0.06-60 ng) was determined using protocol A in the Detailed Protocol section. Assay range: 200 pg-20 ng (1283 Units/mg).

Figure 6: Staurosporine Inhibition of Chk2

The activity of Human, Recombinant Chk2 (6 ng) was determined using protocol A in the Detailed Protocol section in the presence of increasing Staurosporine concentration.

Figure 7: Activity of Chk2 Immunoprecipitated from HeLa Cell Lysate

Near-confluent HeLa cells were treated with UV (50 J/m²) or camptothecin (10 µM) for 2 h. Following exposure to UV the cells were incubated in fresh medium for 2 h at 37°C in a CO₂ incubator. Immunoprecipitation of Chk2 was performed using the indicated antibody and kinase activity was determined according to Detailed Protocol B.