Phospho-Chk1 (S296) Recombinant Rabbit Monoclonal Antibody [SN06-50]

☑ Cell treatment (CT)

Western blot analysis of Phospho-Chk1 (S296) on different lysates with Rabbit anti-Phospho-Chk1 (S296) antibody (ET1611-76) at 1/1,000 dilution.

Lane 1: HEK-293 cell lysate
Lane 2: HEK-293 treated with 200nM Calyculin A for 1 hour cell lysate
Lane 3: HEK-293 treated with 200nM Calyculin A for 1 hour cell lysate, then the membrane treated with λpp for 1 hour
Lane 4: NIH/3T3 cell lysate
Lane 5: NIH/3T3 treated with 100nM Calyculin A for 30 minutes cell lysate
Lane 6: NIH/3T3 treated with 100nM Calyculin A for 30 minutes cell lysate, then the membrane treated with λpp for 1 hour
Lane 7: C6 cell lysate
Lane 8: C6 treated with 100ng/mL Calyculin A for 1 hour cell lysate
Lane 9: C6 treated with 100ng/mL Calyculin A for 1 hour cell lysate, then the membrane treated with λpp for 1 hour

Lysates/proteins at 20 µg/Lane.

Predicted band size: 54 kDa
Observed band size: 54 kDa

Exposure time: Lane 1-3: 20 seconds; Lane 4-9: 3 minutes;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1611-76) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Immunohistochemical analysis of paraffin-embedded human tonsil tissue with Rabbit anti-Phospho-Chk1 (S296) antibody (ET1611-76) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1611-76) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Immunohistochemical analysis of paraffin-embedded human breast cancer tissue with Rabbit anti-Phospho-Chk1 (S296) antibody (ET1611-76) at 1/200 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1611-76) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

☑ Cell treatment (CT)

Immunocytochemistry analysis of HEK-293 cells treated with or without 100nM Calyculin A for 30 minutes labeling Phospho-Chk1 (S296) with Rabbit anti-Phospho-Chk1 (S296) antibody (ET1611-76) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-Chk1 (S296) antibody (ET1611-76) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.