ET1701-76

Occludin Recombinant Rabbit Monoclonal Antibody [JJ091-08]

cat.: ET1701-76

Product Type:	Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity:	Human, Mouse, Rat, Cynomolgus monkey, Pig
Applications:	WB, IP, FC, IF-Cell, IHC-P, IHC-Fr
Clonality:	Monoclonal
Clone number:	JJ091-08

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: 59 kDa
Isotype:	IgG
Immunogen:	Synthetic peptide within N-terminal human Occludin.
Positive control:	Human colon tissue, mouse colon tissue, rat colon tissue, Caco-2, MCF-7 cell lysate, Mouse smooth muscle tissue lysate.
Subcellular location:	Cell membrane, tight junction.
Recommended Dilutions: WB FC IP IF-Cell IHC-P IHC-Fr	1:1,000-1:2,000 1:1,000 Use at an assay dependent concentration. 1:500 1:100 1:500
Uniprot #:	SwissProt: Q16625 Human \| Q61146 Mouse \| Q6P6T5 Rat
Alternative names:	BLCPMG FLJ08163 FLJ18079 FLJ77961 FLJ94056 MGC34277 Occludin Ocln OCLN_HUMAN Tight junction protein occludin

Images

	Fig1: Western blot analysis of Occludin on different lysates with Rabbit anti-Occludin antibody (ET1701-76) at 1/2,000 dilution. Lane 1: HAP1-parental cell lysate Lane 2: HAP1-Occludin KD cell lysate Lysates/proteins at 10 µg/Lane. Predicted band size: 59 kDa Observed band size: 59 kDa Exposure time: 180 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 (ET1701-76) at 1/2,000 dilution was used in 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.
	Fig2: Application: IHC-Fr Species: Mouse Site: Colon Sample: Frozen section Antibody concentration: 1:500 Antigen retrieval: Not required
	Fig3: Immunohistochemical analysis of paraffin-embedded human colon tissue with Rabbit anti-Occludin antibody (ET1701-76) 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 (ET1701-76) 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.

	Fig4: Immunohistochemical analysis of paraffin-embedded mouse colon tissue with Rabbit anti-Occludin antibody (ET1701-76) 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 (ET1701-76) 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.
	Fig5: Immunohistochemical analysis of paraffin-embedded rat colon tissue with Rabbit anti-Occludin antibody (ET1701-76) 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 (ET1701-76) 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.
	Fig6: Immunocytochemistry analysis of Caco-2 cells labeling Occludin with Rabbit anti-Occludin antibody (ET1701-76) at 1/500 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-Occludin antibody (ET1701-76) at 1/500 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.

Fig7: Flow cytometric analysis of Caco-2 cells labeling Occludin.

Cells were fixed and permeabilized. Then stained with the primary antibody (ET1701-76, 1μg/mL) (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: Western blot analysis of Occludin on different lysates with Rabbit anti-Occludin antibody (ET1701-76) at 1/1,000 dilution.

Lane 1: MCF-7 cell lysate (10 µg/Lane)
Lane 2: Mouse smooth muscle tissue lysate (20 µg/Lane)

Predicted band size: 59 kDa
Observed band size: 59 kDa

Exposure time: 2 minutes;

12% 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 (ET1701-76) 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 1:300,000 dilution was used for 1 hour at room temperature.

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