WB
Western blot analysis of Rad21 on different lysates with Rabbit anti-Rad21 antibody at 1/5,000 dilution.
Lane 1: HeLa cell lysate
Lane 2: MCF7 cell lysate
Lane 3: NIH/3T3 cell lysate
Lane 4: RAW264.7 cell lysate
Lane 5: C6 cell lysate
Lysates/proteins at 10 µg/Lane.
Predicted band size: 72 kDa
Observed band size: 120 kDa
Exposure time: 11 seconds; ECL: K1801;
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 1/5,000 dilution was used in primary antibody dilution at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1/50,000 dilution was used for 1 hour at room temperature.
WB
Western blot analysis of Rad21 on different lysates with Rabbit anti-Rad21 antibody at 1/5,000 dilution.
Lane 1: Mouse brain tissue lysate
Lane 2: Rat brain tissue lysate
Lysates/proteins at 20 µg/Lane.
Predicted band size: 72 kDa
Observed band size: 120 kDa
Exposure time: 1 minute 18 seconds; ECL: K1801;
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 1/5,000 dilution was used in primary antibody dilution at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1/50,000 dilution was used for 1 hour at room temperature.
IHC
Immunohistochemical analysis of paraffin-embedded human breast cancer tissue with Rabbit anti-Rad21 antibody at 1/500 dilution.
The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 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 (HA724078) at 1/500 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.
IP
Rad21 was immunoprecipitated from 0.2 mg HeLa cell lysate with at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA724078 at 1/5,000 dilution. HRP Conjugated Anti-Rabbit IgG for IP Nano-secondary antibody at 1/5,000 dilution was used for 1 hour at room temperature.
Lane 1: HeLa cell lysate (input)
Lane 2: IP in HeLa cell lysate
Lane 3: Rabbit IgG instead of in HeLa cell lysate
Blocking/Dilution buffer: primary antibody dilution
Exposure time: 10 seconds; ECL: K1801
ICC/IF
Immunocytochemistry analysis of HeLa cells labeling Rad21 with Rabbit anti-Rad21 antibody at 1/500 dilution.
Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 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-Rad21 antibody at 1/500 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L 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 was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L was used as the secondary antibody at 1/1,000 dilution.