Phospho-Smad2 (S250) Recombinant Rabbit Monoclonal Antibody [SD207-1] - BSA and Azide free

☑ Cell treatment (CT)

Western blot analysis of Phospho-Smad2 (S250) on different lysates with Rabbit anti-Phospho-Smad2 (S250) antibody (HA750291) at 1/5,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: HeLa treated with 200nM PMA for 30 minutes cell lysate
Lane 3: NIH/3T3 cell lysate
Lane 4: NIH/3T3 treated with 200nM PMA for 30 minutes cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 52 kDa
Observed band size: 55 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 (HA750291) 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.

☑ Cell treatment (CT)

Western blot analysis of Phospho-Smad2 (S250) on different lysates with Rabbit anti-Phospho-Smad2 (S250) antibody (HA750291) at 1/5,000 dilution.

Lane 1: C6 cell lysate
Lane 2: C6 treated with 200nM PMA for 30 minutes cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 52 kDa
Observed band size: 55 kDa

Exposure time: 2 minutes; 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 (HA750291) 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.

☑ Cell treatment (CT)

Phospho-Smad2 (S250) was immunoprecipitated from 0.2 mg HeLa treated with 100nM TPA for 30 minutes cell lysate with HA750291 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA750291 at 1/5,000 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: HeLa treated with 100nM TPA for 30 minutes cell lysate (input)
Lane 2: HA750291 IP in HeLa treated with 100nM TPA for 30 minutes cell lysate
Lane 3: Rabbit IgG instead of HA750291 in HeLa treated with 100nM TPA for 30 minutes cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 35 seconds; ECL: K1802

Immunohistochemical analysis of paraffin-embedded human lung cancer tissue with Rabbit anti-Phospho-Smad2 (S250) antibody (HA750291) at 1/50 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 (HA750291) at 1/50 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 testis tissue with Rabbit anti-Phospho-Smad2 (S250) antibody (HA750291) at 1/50 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 (HA750291) at 1/50 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 testis tissue with Rabbit anti-Phospho-Smad2 (S250) antibody (HA750291) at 1/50 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 (HA750291) at 1/50 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 HeLa cells labeling Phospho-Smad2 (S250).

Cells were fixed and permeabilized. Then stained with the primary antibody (HA750291, 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).