p38 alpha Recombinant Rabbit Monoclonal Antibody [JF55-07]

Western blot analysis of p38 alpha on different lysates with Rabbit anti-p38 alpha antibody (ET1702-65) at 1/5,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: Jurkat cell lysate
Lane 3: HEK-293 cell lysate
Lane 4: NIH/3T3 cell lysate
Lane 5: Neuro-2a cell lysate
Lane 6: RAW264.7 cell lysate
Lane 7: PC-12 cell lysate
Lane 8: C6 cell lysate

Lysates/proteins at 15 µg/Lane.

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

Exposure time: 3 minutes;

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 (ET1702-65) 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.

☑ Knockout (KO)

All lanes: Western blot analysis of p38 alpha with anti-p38 alpha antibody[JF55-07] (ET1702-65) at 1:500 dilution.
Lane 1: Wild-type Hela whole cell lysate (10 µg).
Lane 2: p38 alpha knockout Hela whole cell lysate (10 µg).

ET1702-65 was shown to specifically react with p38 alpha in wild-type Hela cells. No band was observed when p38 alpha knockout sample was tested. Wild-type and p38 alpha knockout samples were subjected to SDS-PAGE. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM in TBST for 1 hour at room temperature. The primary antibody (ET1702-65, 1/500) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG-HRP Secondary Antibody (HA1001) at 1:100,000 dilution was used for 1 hour at room temperature.

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

Lane 1: THP-1 cell lysate (10 µg/Lane)
Lane 2: C2C12 cell lysate (10 µg/Lane)
Lane 3: PC-12 cell lysate (10 µg/Lane)
Lane 4: Rat kidney tissue lysate (20 µg/Lane)
Lane 5: Mouse kidney tissue lysate (20 µg/Lane)

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

Exposure time: 3 minutes 10 seconds;

10% 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 (ET1702-65) at 1/1,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:100,000 dilution was used for 1 hour at room temperature.

Immunocytochemistry analysis of RAW264.7 cells labeling p38 alpha with Rabbit anti-p38 alpha antibody (ET1702-65) 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-p38 alpha antibody (ET1702-65) 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.

Immunocytochemistry analysis of PC-12 cells labeling p38 alpha with Rabbit anti-p38 alpha antibody (ET1702-65) 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-p38 alpha antibody (ET1702-65) 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 RAW264.7 cells labeling p38 alpha.

Cells were fixed and permeabilized. Then stained with the primary antibody (ET1702-65, 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).

Flow cytometric analysis of HeLa cells labeling p38 alpha.

Cells were fixed and permeabilized. Then stained with the primary antibody (ET1702-65, 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).

Immunohistochemical analysis of paraffin-embedded mouse heart tissue with Rabbit anti-p38 alpha antibody (ET1702-65) at 1/200 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 (ET1702-65) at 1/200 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.