Phospho-p38 alpha (T180 + Y182) Recombinant Rabbit Monoclonal Antibody [PSH06-35]

☑ Cell treatment (CT)

Western blot analysis of Phospho-p38 alpha (T180 + Y182) on different lysates with Rabbit anti-Phospho-p38 alpha (T180 + Y182) antibody (HA722669) at 1/2,000 dilution and p38 alpha antibody (ET1702-65) at 1/1,000 dilution.

Lane 1: Jurkat cell lysate (20 µg/Lane)
Lane 2: Jurkat treated with UV for 1 hour cell lysate (20 µg/Lane)
Lane 3: NIH/3T3 cell lysate (20 µg/Lane)
Lane 4: NIH/3T3 treated with 25μg/mL Anisomycin for 30 minutes cell lysate (20 µg/Lane)
Lane 5: C6 cell lysate (20 µg/Lane)
Lane 6: C6 treated with 25μg/mL Anisomycin for 30 minutes cell lysate (20 µg/Lane)

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

Exposure time: 3 minutes; 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 (HA722669) at 1/2,000 dilution and p38 alpha 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.

☑ Cell treatment (CT)

Immunocytochemistry analysis of Jurkat cells treated with UV for 1 hour labeling Phospho-p38 alpha (T180 + Y182) with Rabbit anti-Phospho-p38 alpha (T180 + Y182) antibody (HA722669) at 1/50 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 + Y182) antibody (HA722669) at 1/50 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)

Immunocytochemistry analysis of NIH/3T3 cells treated with 25μg/mL Anisomycin for 30 minutes labeling Phospho-p38 alpha (T180 + Y182) with Rabbit anti-Phospho-p38 alpha (T180 + Y182) antibody (HA722669) 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 + Y182) antibody (HA722669) 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.

Flow cytometric analysis of Jurkat cells untreated (left) or treated (right) with UV for 1 hour labeling Phospho-p38 alpha (T180 + Y182).

Cells were fixed and permeabilized. Then stained with the primary antibody (HA722669, 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).