Cyclin D1 Recombinant Rabbit Monoclonal Antibody [SA38-08]

☑ Relative expression (RE)

Western blot analysis of Cyclin D1 on different lysates with Rabbit anti-Cyclin D1 antibody (ET1601-31) at 1/5,000 dilution and competitor's antibody at 1/5,000 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.

Predicted band size: 34 kDa
Observed band size: 35 kDa

Exposure time: 20 seconds; ECL: K1802;
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 (ET1601-31) at 1/5,000 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 (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

☑ Knockdown (KD)

Western blot analysis of Cyclin D1 on different lysates with Rabbit anti-Cyclin D1 antibody (ET1601-31) at 1/5,000 dilution.

Lane 1: MCF7-si NT cell lysate
Lane 2: MCF7-si Cyclin D1 cell lysate

Lysates/proteins at 10 µg/Lane.

Predicted band size: 34 kDa
Observed band size: 34 kDa

Exposure time: 17 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 (ET1601-31) at 1/5,000 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.

Immunocytochemistry analysis of Neuro-2a cells labeling Cyclin D1 with Rabbit anti-Cyclin D1 antibody (ET1601-31) at 1/2,000 dilution and competitor's antibody at 1/1,600 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-Cyclin D1 antibody (ET1601-31) at 1/2,000 dilution and competitor's antibody at 1/1,600 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.

Flow cytometric analysis of MCF7 cells labeling Cyclin D1.

Cells were fixed and permeabilized. Then stained with the primary antibody (ET1601-31, red) at 1/5,000 dilution and competitor's antibody (red) at 1/2,000 dilution, compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

Immunohistochemical analysis of paraffin-embedded human colon cancer tissue with Rabbit anti-Cyclin D1 antibody (ET1601-31) at 1/1,000 dilution.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins. The section was incubated with ET1601-31 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Immunohistochemical analysis of paraffin-embedded human tonsil tissue using anti-Cyclin D1 antibody. 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 5% BSA for 30 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1601-31, 1/200) for 30 minutes 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 colon carcinoma tissue using anti-Cyclin D1 antibody. 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 5% BSA for 30 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1601-31, 1/200) for 30 minutes 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 small intestine tissue using anti-Cyclin D1 antibody. 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 5% BSA for 30 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1601-31, 1/200) for 30 minutes 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 colon carcinoma tissue using anti-Cyclin D1 antibody. 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 5% BSA for 30 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1601-31, 1/200) for 30 minutes 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 mouse stomach tissue with Rabbit anti-Cyclin D1 antibody (ET1601-31) 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 (ET1601-31) 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 rat esophagus tissue with Rabbit anti-Cyclin D1 antibody (ET1601-31) 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 (ET1601-31) 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.

Application: IF-Tissue

Species: Human

Site: colon cancer

Sample: Paraffin-embedded section

Antibody concentration: 1/500

☑ Relative expression (RE)

Immunocytochemistry analysis of SH-SY5Y (positive) and 293T (negative) labeling Cyclin D1 with Rabbit anti-Cyclin D1 antibody (ET1601-31) at 1/2,000 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-Cyclin D1 antibody (ET1601-31) at 1/2,000 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.

Immunocytochemistry analysis of C6 cells labeling Cyclin D1 with Rabbit anti-Cyclin D1 antibody (ET1601-31) at 1/2,000 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-Cyclin D1 antibody (ET1601-31) at 1/2,000 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.

Cyclin D1 was immunoprecipitated from 0.2 mg MCF7 cell lysate with ET1601-31 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using ET1601-31 at 1/1,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: MCF7 cell lysate (input)
Lane 2: ET1601-31 IP in MCF7 cell lysate
Lane 3: Rabbit IgG instead of ET1601-31 in MCF7 cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 5 seconds; ECL: K1802

Immunohistochemical analysis of paraffin embedded human colon cancer tissue using anti-Cyclin D1 antibody (1/1,000) performed on the Leica® BOND™ RX.