Phospho-p38 alpha (T180) Recombinant Rabbit Monoclonal Antibody [PSH06-25]

☑ Cell treatment (CT)

Western blot analysis of Phospho-p38 alpha (T180) on different lysates with Rabbit anti-Phospho-p38 alpha (T180) antibody (HA722657) at 1/2,000 dilution and pan p38 antibody (ET1702-65) at 1/1,000 dilution.

Lane 1: Jurkat cell lysate
Lane 2: Jurkat treated with UV for 1 hour cell lysate
Lane 3: NIH/3T3 cell lysate
Lane 4: NIH/3T3 treated with 25μg/mL Anisomycin for 30 minutes cell lysate
Lane 5: C6 cell lysate
Lane 6: C6 treated with 25μg/mL Anisomycin for 30 minutes cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 41 kDa
Observed band size: 38 kDa

Exposure time: 1 minute 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 (HA722657) at 1/2,000 dilution and pan p38 antibody (ET1702-65) at 1/1,000 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.

Immunohistochemical analysis of paraffin-embedded mouse colon tissue with Rabbit anti-Phospho-p38 alpha (T180) antibody (HA722657) at 1/500 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) 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 (HA722657) at 1/500 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 colon tissue with Rabbit anti-Phospho-p38 alpha (T180) antibody (HA722657) at 1/500 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) 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 (HA722657) at 1/500 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.

☑ Cell treatment (CT)

Immunocytochemistry analysis of NIH/3T3 cells treated with 25μg/mL Anisomycin for 30 minutes labeling Phospho-p38 alpha (T180) with Rabbit anti-Phospho-p38 alpha (T180) antibody (HA722657) 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-Phospho-p38 alpha (T180) antibody (HA722657) 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. 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)

Flow cytometric analysis of NIH/3T3 cells untreated / treated with 25μg/mL Anisomycin for 30 minutes labeling Phospho-p38 alpha (T180).

Cells were fixed and permeabilized. Then stained with the primary antibody (HA722657, 1/20,000) (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).