eNOS Recombinant Rabbit Monoclonal Antibody [PSH16-96]
cat.: HA751610
Product Type: Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity: Human, Mouse, Rat
Applications: WB, IF-Cell, IHC-P, FC, IP
Clonality: Monoclonal
Clone number: PSH16-96
Form: Liquid
Storage condition: Store at +4℃ after thawing. Aliquot store at -20℃. Avoid repeated freeze / thaw cycles.
Storage buffer: 1*PBS (pH7.4).
Concentration: 1ug/ul
Purification: Protein A affinity purified.
Molecular weight: Predicted band size: 133 kDa
Isotype: IgG
Immunogen: Recombinant protein within human eNOS aa 950-1,203.
Positive control: EA.hy926 cell lysate, bEnd.3 cell lysate, Mouse placenta tissue lysate, Mouse kidney tissue lysate, Rat placenta tissue lysate, Rat kidney tissue lysate, EA.hy926, human colon carcinoma tissue, human placenta tissue, human kidney tissue, human spleen tissue, mouse spleen tissue, rat kidney tissue, rat spleen tissue, rat placenta tissue.
Subcellular location: Cell membrane, Membrane, caveola, Cytoplasm, cytoskeleton, Golgi apparatus.
Recommended Dilutions:
  WB
  IF-Cell
  IHC-P
  FC
  IP
1:5,000
1:50
1:50-1:200
1:1,000
1-2μg/sample
Uniprot #: SwissProt: P29474 Human | P70313 Mouse | Q62600 Rat
Alternative names: cNOS Constitutive NOS EC NOS EC-NOS ecNOS Endothelial nitric oxidase synthase Endothelial nitric oxide synthase Endothelial nitric oxide synthase 3 Endothelial NOS eNOS Nitric oxide synthase 3 (endothelial cell) Nitric oxide synthase 3 Nitric oxide synthase 3 endothelial cell Nitric oxide synthase endothelial Nitric oxide synthase, endothelial NOS 3 NOS III NOS type III NOS3 NOS3_HUMAN NOSIII
Images
HA751610_1.jpg Fig1: Western blot analysis of eNOS on different lysates with Rabbit anti-eNOS antibody (HA751610) at 1/5,000 dilution.

Lane 1: EA.hy926 cell lysate (20 µg/Lane)
Lane 2: HeLa cell lysate (negative) (20 µg/Lane)
Lane 3: bEnd.3 cell lysate (20 µg/Lane)
Lane 4: RAW264.7 cell lysate (negative) (20 µg/Lane)
Lane 5: Mouse placenta tissue lysate (30 µg/Lane)
Lane 6: Mouse kidney tissue lysate (30 µg/Lane)
Lane 7: Rat placenta tissue lysate (30 µg/Lane)
Lane 8: Rat kidney tissue lysate (30 µg/Lane)

Predicted band size: 133 kDa
Observed band size: 133 kDa

Exposure time: Lane 1-2: 10 seconds; Lane 3-8: 3 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 (HA751610) at 1/5,000 dilution was used in primary antibody dilution (K1803) at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
HA751610_2.jpg Fig2: Immunocytochemistry analysis of EA.hy926 (positive) and HeLa (negative) labeling eNOS with Rabbit anti-eNOS antibody (HA751610) at 1/50 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 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-eNOS antibody (HA751610) 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 (HA601187, 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.
HA751610_3.jpg Fig3: Immunohistochemical analysis of paraffin-embedded human colon carcinoma tissue with Rabbit anti-eNOS antibody (HA751610) at 1/200 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 (HA751610) 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.
HA751610_4.jpg Fig4: Immunohistochemical analysis of paraffin-embedded human placenta tissue with Rabbit anti-eNOS antibody (HA751610) at 1/200 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 (HA751610) 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.
HA751610_5.jpg Fig5: Immunohistochemical analysis of paraffin-embedded human kidney tissue with Rabbit anti-eNOS antibody (HA751610) 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 (HA751610) 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.
HA751610_6.jpg Fig6: Immunohistochemical analysis of paraffin-embedded human spleen tissue with Rabbit anti-eNOS antibody (HA751610) 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 (HA751610) 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.
HA751610_7.jpg Fig7: Immunohistochemical analysis of paraffin-embedded mouse spleen tissue with Rabbit anti-eNOS antibody (HA751610) 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 (HA751610) 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.
HA751610_8.jpg Fig8: Immunohistochemical analysis of paraffin-embedded rat kidney tissue with Rabbit anti-eNOS antibody (HA751610) 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 (HA751610) 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.
HA751610_9.jpg Fig9: Immunohistochemical analysis of paraffin-embedded rat spleen tissue with Rabbit anti-eNOS antibody (HA751610) 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 (HA751610) 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.
HA751610_10.jpg Fig10: Immunohistochemical analysis of paraffin-embedded rat placenta tissue with Rabbit anti-eNOS antibody (HA751610) at 1/200 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 (HA751610) 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.
HA751610_11.jpg Fig11: Flow cytometric analysis of HeLa (left, negative) and EA.hy926 (right, positive) cells labeling eNOS.

Cells were fixed and permeabilized. Then stained with the primary antibody (HA751610, 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).
HA751610_12.jpg Fig12: Western blot analysis of eNOS on different lysates with Rabbit anti-eNOS antibody (HA751610) at 1/5,000 dilution.

Lane 1: His-tagged eNOS recombinant protein
Lane 2: His-tagged nNOS recombinant protein

Lysates/proteins at 20 ng/Lane.

Exposure time: 2 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 (HA751610) at 1/5,000 dilution was used in primary antibody dilution (K1803) at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
HA751610_13.jpg Fig13: eNOS was immunoprecipitated from 0.2 mg bEnd.3 cell lysate with HA751610 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA751610 at 1/5,000 dilution. HRP Conjugated Anti-Rabbit IgG for IP Nano-secondary antibody at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: bEnd.3 cell lysate (input)
Lane 2: HA751610 IP in bEnd.3 cell lysate
Lane 3: Rabbit IgG instead of HA751610 in bEnd.3 cell lysate

Blocking/Dilution buffer: primary antibody dilution (K1803)
Exposure time: 3 minutes; ECL: K1801
Note: All products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”.