Phospho-MCM2 (S41) Recombinant Rabbit Monoclonal Antibody [JE50-04]

Western blot analysis of Phospho-MCM2 (S41) on different lysates with Rabbit anti-Phospho-MCM2 (S41) antibody (HA722279) at 1/1,000 dilution.

Lane 1: A431 cell lysate (20 µg/Lane)
Lane 2: HeLa cell lysate (20 µg/Lane)
Lane 3: HEK-293 cell lysate (20 µg/Lane)
Lane 4: THP-1 cell lysate (20 µg/Lane)
Lane 5: Mouse spleen tissue lysate (30 µg/Lane)
Lane 6: Mouse thymus tissue lysate (30 µg/Lane)
Lane 7: Rat spleen tissue lysate (30 µg/Lane)
Lane 8: Rat thymus tissue lysate (30 µg/Lane)

Predicted band size: 102 kDa
Observed band size: 130 kDa

Exposure time: Lane 1-4: 8 seconds; Lane 5-8: 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 (HA722279) at 1/1,000 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 THP-1 cells labeling Phospho-MCM2 (S41) with Rabbit anti-Phospho-MCM2 (S41) antibody (HA722279) at 1/500 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-Phospho-MCM2 (S41) antibody (HA722279) at 1/500 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.

☑ Cell treatment (CT)

Immunohistochemical analysis of paraffin-embedded human tonsil tissue untreated / treated with λpp with Rabbit anti-Phospho-MCM2 (S41) antibody (HA722279) 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 (HA722279) 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 human spleen tissue with Rabbit anti-Phospho-MCM2 (S41) antibody (HA722279) 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 (HA722279) 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 mouse spleen tissue with Rabbit anti-Phospho-MCM2 (S41) antibody (HA722279) 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 (HA722279) 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 spleen tissue with Rabbit anti-Phospho-MCM2 (S41) antibody (HA722279) 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 (HA722279) 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.

Flow cytometric analysis of THP-1 cells labeling Phospho-MCM2 (S41).

Cells were fixed and permeabilized. Then stained with the primary antibody (HA722279, 1/1,000) (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).

Phospho-MCM2 (S41) was immunoprecipitated from 0.2 mg THP-1 cell lysate with HA722279 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA722279 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: THP-1 cell lysate (input)
Lane 2: HA722279 IP in THP-1 cell lysate
Lane 3: Rabbit IgG instead of HA722279 in THP-1 cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 2 minutes 5 seconds; ECL: K1801