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Western blot analysis of SIRT3 on different lysates with Rabbit anti-SIRT3 antibody (HA722251) at 1/2,000 dilution and competitor's antibody at 1/2,000 dilution.
Lane 1: PANC-1 cell lysate
Lane 2: HepG2 cell lysate
Lane 3: A549 cell lysate
Lane 4: SiHa cell lysate
Lane 5: Mouse liver tissue lysate
Lane 6: Mouse kidney tissue lysate
Lane 7: Rat liver tissue lysate
Lysates/proteins at 20 µg/Lane.
Predicted band size: 44 kDa
Observed band size: 28 kDa
Exposure time: 30 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 (HA722251) at 1/2,000 dilution and competitor's antibody at 1/2,000 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.
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Immunohistochemical analysis of paraffin-embedded human liver tissue with Rabbit anti-SIRT3 antibody (HA722251) at 1/1,000 dilution and competitor's antibody at 1/1,000 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 (HA722251) at 1/1,000 dilution and competitor's antibody 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.
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Immunohistochemical analysis of paraffin-embedded human colon tissue with Rabbit anti-SIRT3 antibody (HA722251) at 1/1,000 dilution and competitor's antibody at 1/1,000 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 (HA722251) at 1/1,000 dilution and competitor's antibody 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.
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Western blot analysis of SIRT3 on different lysates with Rabbit anti-SIRT3 antibody (HA722251) at 1/2,000 dilution.
Lane 1: HeLa cell lysate (20 µg/Lane)
Lane 2: HEK-293 cell lysate (20 µg/Lane)
Lane 3: PANC-1 cell lysate (20 µg/Lane)
Lane 4: HepG2 cell lysate (20 µg/Lane)
Lane 5: SW480 cell lysate (20 µg/Lane)
Lane 6: A549 cell lysate (20 µg/Lane)
Lane 7: HT-29 cell lysate (20 µg/Lane)
Lane 8: SiHa cell lysate (20 µg/Lane)
Lane 9: Mouse liver tissue lysate (40 µg/Lane)
Lane 10: Mouse kidney tissue lysate (40 µg/Lane)
Lane 11: Rat liver tissue lysate (40 µg/Lane)
Lane 12: Rat kidney tissue lysate (40 µg/Lane)
Predicted band size: 44 kDa
Observed band size: 28 kDa
Exposure time: 25 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 (HA722251) at 1/2,000 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.
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Immunohistochemical analysis of paraffin-embedded human kidney tissue with Rabbit anti-SIRT3 antibody (HA722251) at 1/1,000 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 (HA722251) 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.
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Immunohistochemical analysis of paraffin-embedded mouse liver tissue with Rabbit anti-SIRT3 antibody (HA722251) at 1/1,000 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 (HA722251) 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.
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Immunohistochemical analysis of paraffin-embedded rat liver tissue with Rabbit anti-SIRT3 antibody (HA722251) at 1/1,000 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 (HA722251) 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.
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Immunocytochemistry analysis of HepG2 cells labeling SIRT3 with Rabbit anti-SIRT3 antibody (HA722251) 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-SIRT3 antibody (HA722251) 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. Counterstained with Mitotracker (Red). Nuclear DNA was labelled in blue with DAPI.
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Flow cytometric analysis of HepG2 cells labeling SIRT3.
Cells were fixed and permeabilized. Then stained with the primary antibody (HA722251, 1μg/mL) (red) 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).
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Flow cytometric analysis of NIH/3T3 cells labeling SIRT3.
Cells were fixed and permeabilized. Then stained with the primary antibody (HA722251, 1μg/mL) (red) 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).
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SIRT3 was immunoprecipitated from 0.2 mg HepG2 cell lysate with HA722251 at 2 µg/25 µl agarose. Western blot was performed from the immunoprecipitate using HA722251 at 1/2,000 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: HepG2 cell lysate (input)
Lane 2: HA722251 IP in HepG2 cell lysate
Lane 3: Rabbit IgG instead of HA722251 in HepG2 cell lysate
Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 1 minute 17 seconds; ECL: K1801
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