ZO3 Recombinant Rabbit Monoclonal Antibody [PSH19-98]
cat.: HA724118
Product Type: Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity: Human, Mouse, Rat
Applications: WB, IHC-P, IF-Cell, FC, IP
Clonality: Monoclonal
Clone number: PSH19-98
Form: Liquid
Storage condition: Shipped at 4℃. Store at +4℃ short term (1-2 weeks). Store at -20℃ long term.
Storage buffer: 1*PBS (pH7.4), 0.1% BSA, 40% Glycerol, 0.2% Proclean 950.
Concentration: 1ug/ul
Purification: Protein A affinity purified.
Molecular weight: Predicted band size: 101 kDa
Isotype: IgG
Immunogen: Recombinant protein within human ZO3 aa 1-380.
Positive control: MCF7 cell lysate, HT-29 cell lysate, Caco-2 cell lysate, human testis tissue, human small intestine tissue, mouse testis tissue, mouse small intestine tissue, rat testis tissue, rat small intestine tissue, MCF7.
Subcellular location: Cell membrane, Cell junction, tight junction, Nucleus.
Recommended Dilutions:
  WB
  IHC-P
  IF-Cell
  FC
  IP
1:5,000
1:100
1:100
1:1,000
1-2μg/sample
Uniprot #: SwissProt: O95049 Human | Q9QXY1 Mouse
Entrez Gene: 314640 Rat
Alternative names: Tight junction protein 3 Tight junction protein ZO-3 Tight junction protein ZO3 TJP3 ZO3 ZO3_HUMAN Zona occludens protein 3 Zonula occludens protein 3
Images
HA724118_1.jpg Fig1: Western blot analysis of ZO3 on different lysates with Rabbit anti-ZO3 antibody (HA724118) at 1/5,000 dilution.

Lane 1: MCF7 cell lysate
Lane 2: HT-29 cell lysate
Lane 3: Caco-2 cell lysate

Lysates/proteins at 10 µg/Lane.

Predicted band size: 101 kDa
Observed band size: 150 kDa

Exposure time: 30 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 (HA724118) 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.
HA724118_2.jpg Fig2: Immunohistochemical analysis of paraffin-embedded human testis tissue with Rabbit anti-ZO3 antibody (HA724118) at 1/100 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 (HA724118) at 1/100 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.
HA724118_3.jpg Fig3: Immunohistochemical analysis of paraffin-embedded human small intestine tissue with Rabbit anti-ZO3 antibody (HA724118) at 1/100 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 (HA724118) at 1/100 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.
HA724118_4.jpg Fig4: Immunohistochemical analysis of paraffin-embedded human skeletal muscle tissue (negative) with Rabbit anti-ZO3 antibody (HA724118) 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 (HA724118) 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.
HA724118_5.jpg Fig5: Immunohistochemical analysis of paraffin-embedded mouse testis tissue with Rabbit anti-ZO3 antibody (HA724118) at 1/100 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 (HA724118) at 1/100 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.
HA724118_6.jpg Fig6: Immunohistochemical analysis of paraffin-embedded mouse small intestine tissue with Rabbit anti-ZO3 antibody (HA724118) at 1/100 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 (HA724118) at 1/100 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.
HA724118_7.jpg Fig7: Immunohistochemical analysis of paraffin-embedded mouse skeletal muscle tissue (negative) with Rabbit anti-ZO3 antibody (HA724118) 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 (HA724118) 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.
HA724118_8.jpg Fig8: Immunohistochemical analysis of paraffin-embedded rat testis tissue with Rabbit anti-ZO3 antibody (HA724118) at 1/100 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 (HA724118) at 1/100 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.
HA724118_9.jpg Fig9: Immunohistochemical analysis of paraffin-embedded rat small intestine tissue with Rabbit anti-ZO3 antibody (HA724118) at 1/100 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 (HA724118) at 1/100 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.
HA724118_10.jpg Fig10: Immunohistochemical analysis of paraffin-embedded rat skeletal muscle tissue (negative) with Rabbit anti-ZO3 antibody (HA724118) 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 (HA724118) 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.
HA724118_11.jpg Fig11: Immunocytochemistry analysis of MCF7 cells labeling ZO3 with Rabbit anti-ZO3 antibody (HA724118) at 1/100 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-ZO3 antibody (HA724118) 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 (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.
HA724118_12.jpg Fig12: Flow cytometric analysis of MCF7 cells labeling ZO3.

Cells were fixed and permeabilized. Then stained with the primary antibody (HA724118, 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).
HA724118_13.jpg Fig13: ZO3 was immunoprecipitated from 0.2 mg MCF7 cell lysate with HA724118 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA724118 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: MCF7 cell lysate (input)
Lane 2: HA724118 IP in MCF7 cell lysate
Lane 3: Rabbit IgG instead of HA724118 in MCF7 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”.