WB
Western blot analysis of Cyclin D1 on different lysates with Rabbit anti-Cyclin D1 antibody at 1/5000 dilution and competitor's antibody at 1/5000 dilution.
Lane 1: MCF7 cell lysate,
Lane 2: K-562 cell lysate (negative),
Lane 3: A431 cell lysate,
Lane 4: Neuro-2a cell lysate,
Lane 5: NIH/3T3 cell lysate,
Lane 6: C6 cell lysate,
Lane 7: SH-SY5Y cell lysate,
Lysates/proteins at 20 µg/Lane.
Exposure time: 20 seconds;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 1/5000 dilution and competitor's antibody at 1/5,000 dilution were used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1/50000 dilution was used for 1 hour at room temperature.
WB
Western blot analysis of Cyclin D1 on different lysates with Rabbit anti-Cyclin D1 antibody
at 1/5000 dilution.
Lane 1: MCF7-si NT cell lysate,
Lane 2: MCF7-si Cyclin D1 cell lysate,
Lysates/proteins at 10 µg/Lane.
Exposure time: 17 seconds;
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 1/5000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1/50000 dilution was used for 1 hour at room temperature.
ICC/IF
Immunocytochemistry analysis of NIH/3T3 cells labeling Cyclin D1 with Rabbit anti-Cyclin D1 antibody at 1/50 dilution.
Cells were fixed in 4% paraformaldehyde for 10 minutes at 37 ℃, permeabilized with 0.05% Triton X-100 in PBS for 20 minutes, and then blocked with 2% negative goat serum for 30 minutes at room temperature. Cells were then incubated with Rabbit anti-Cyclin D1 antibody at 1/50 dilution in 2% negative goat serum overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 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.
IHC
Immunohistochemical analysis of paraffin-embedded human colon carcinoma tissue with Rabbit anti-Cyclin D1 antibody 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 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.