WB
Western blot analysis of Phospho-Chk1 (S345) on different lysates with Rabbit anti-Phospho-Chk1 (S345) antibody at 1/1,000 dilution.
Lane 1: NIH/3T3 cell lysate,
Lane 2: NIH/3T3 treated with UV for 20 minutes then recover for 2 hours cell lysate,
Lysates/proteins at 20 µg/Lane.
Exposure time: 1 minute;
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 at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1/50,000 dilution was used for 1 hour at room temperature.
ICC/IF
Immunocytochemistry analysis of HeLa cells treated with or without 4mM Hydroxyurea for 20 hours labeling Phospho-Chk1 (S345) with Rabbit anti-Phospho-Chk1 (S345) antibody at 1/200 dilution. Image shown an increased nuclear staining upon Hydroxyurea treatment.
Cells were fixed in 4% paraformaldehyde for 20 minutes at 37 ℃, permeabilized with 0.1% Triton X-100 in PBS permeabilization for 5 minutes, and then blocked with 2% negative goat serum for 60 minutes at room temperature. Cells were then incubated with Rabbit anti-Phospho-Chk1 (S345) antibody at 1/200 dilution in 1% BSA overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (488) 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.