ET1702-65

p38 alpha Recombinant Rabbit Monoclonal Antibody [JF55-07]

cat.: ET1702-65

Product Type:	Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity:	Human, Mouse, Rat
Applications:	WB, IF-Cell, IF-Tissue, IHC-P, FC
Clonality:	Monoclonal
Clone number:	JF55-07

Form:	Liquid
Storage condition:	Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. Avoid repeated freeze / thaw cycles.
Storage buffer:	1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Concentration:	1ug/ul
Purification:	Protein A affinity purified.
Molecular weight:	Predicted band size: 41 kDa
Isotype:	IgG
Immunogen:	Synthetic peptide within C-terminal human MAPK14.
Positive control:	HeLa cell lysate, Jurkat cell lysate, HEK-293 cell lysate, NIH/3T3 cell lysate, Neuro-2a cell lysate, RAW264.7 cell lysate, PC-12 cell lysate, C6 cell lysate, THP-1 cell lysate, C2C12 cell lysate, Rat kidney tissue lysate, Mouse kidney tissue lysate, RAW264.7, PC-12, HeLa, mouse heart tissue.
Subcellular location:	Nucleus, Cytoplasm.
Recommended Dilutions: WB IF-Cell IF-Tissue IHC-P FC	1:2,000-1:5,000 1:100 1:50-1:200 1:50-1:200 1:1,000
Uniprot #:	SwissProt: Q16539 Human \| P47811 Mouse \| P70618 Rat
Alternative names:	CSAID-binding protein Csaids binding protein CSBP CSBP1 CSBP2 CSPB1 Cytokine suppressive anti-inflammatory drug-binding protein EXIP MAP kinase 14 MAP kinase MXI2 MAP kinase p38 alpha MAPK 14 MAPK14 MAX-interacting protein 2 Mitogen-activated protein kinase 14 Mitogen-activated protein kinase p38 alpha MK14_HUMAN Mxi2 p38 p38 MAP kinase p38 mitogen activated protein kinase p38ALPHA p38alpha Exip PRKM14 PRKM15 RK SAPK2A Stress-activated protein kinase 2a

Images

Fig1: 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.

Fig2: 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.

Fig3: 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.

Fig4: 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.

	Fig5: 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.
	Fig6: 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).
	Fig7: 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).

Fig8: 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.

Note: All products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”.