Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply Rabbit monoclonal [EPR20992] to Occludin Suitable for: WB, IHC-P, ICC/IF, IP, Flow Cyt (Intra) Knockout validated Reacts with: Mouse, Rat, Dog, Human, Recombinant fragment Recombinant fragment. This information is proprietary to Abcam and/or its suppliers. WB: Human colon lysate; MDCK, PC-3, HEK-293 and Caco-2 whole cell lysates; Rat brain lysate; Mouse brain and uterus lysates.IHC-P: Human colon and breast tissues; Mouse and rat kidney tissues.ICC/IF: Caco-2 and MDCK cells.Flow Cyt (intra): Caco-2 cells.IP: Caco-2 whole cell lysate. 常规说明 This product is a recombinant monoclonal antibody, which offers several advantages including:- High batch-to-batch consistency and reproducibility- Improved sensitivity and specificity- Long-term security of supply- Animal-free productionFor more information see here.Our RabMAb technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb patents. 存放说明 Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle. 存储溶液 pH: 7.2Preservative: 0.01% Sodium azideConstituents: 0.05% BSA, 40% Glycerol, PBS The Abpromise guarantee Abpromise™承诺保证使用ab216327于以下的经测试应用 \"应用说明”部分 下显示的仅为推荐的起始稀释度；实际最佳的稀释度/浓度应由使用者检定。 1/1000. Detects a band of approximately 65, 53, 25, 23 kDa (predicted molecular weight: 59 kDa). IHC-P (1) 1/200. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. All lanes : Anti-Occludin antibody [EPR20992] (ab216327) at 1/1000 dilutionLane 1 : Wild-type HAP1 whole cell lysate at 40 µgLane 2 : OCLN (Occludin) knockout HAP1 whole cell lysate at 40 µgLane 3 : HeLa whole cell lysate (Low Occludin expression) at 20 µgLane 4 : HepG2 whole cell lysate lysate (High Occludin expression) at 20 µgPredicted band size: 59 kDaLanes 1 - 4: Merged signal (red and green). Green - ab216327 observed at 59 kDa. Red - loading control, ab9484, observed at 37 kDa.ab216327 was shown to recognize Occludin in wild-type HAP1 cells as signal was lost at the expected MW in OCLN (Occludin) knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and OCLN (Occludin) knockout samples were subjected to SDS-PAGE. ab216327 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4 C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H L (IRDye 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H L (IRDye 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.Occludin expression in HeLa is expected to be negative. Immunohistochemical analysis of paraffin-embedded human colon tissue labeling Occludin with ab216327 at 1/200 dilution, followed by Goat Anti-Rabbit IgG H L (HRP) ready to use. Membranous staining on human colon is observed (PMID: 24268521). Counter stained with Hematoxylin.Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H L (HRP) ready to use.Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. Lane 1 : Anti-Occludin antibody [EPR8208] (ab167161) at 1/5000 dilutionLane 2 : Anti-Occludin antibody (ab31721) at 1/1000 dilutionLane 3 : Anti-Occludin antibody [EPR20992] (ab216327) at 1/5000 dilutionAll lanes : Recombinant Human Occludin protein (ab114189)Lysates/proteins at 0.025 µg per lane.SecondaryAll lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilutionPredicted band size: 59 kDaObserved band size: 85 kDa why is the actual band size different from the predicted?Blocking and diluting buffer: 5% NFDM/TBSTExposure time: 5.5 seconds Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Caco-2 (human colorectal adenocarcinoma cell line) cells labelingOccludin with ab216327 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H L (Alexa Fluor 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing membrane staining on Caco-2 cell line.The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor 594) (ab195889) at 1/200 dilution (red).Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H L (Alexa Fluor 488) (ab150077) secondary antibody at 1/1000 dilution. All lanes : Anti-Occludin antibody [EPR20992] (ab216327) at 1/1000 dilutionLane 1 : Human colon lysate at 20 µgLane 2 : MDCK (canine kidney cell line) cell lysate at 20 µgLane 3 : PC-3 (human prostate adenocarcinoma cell line) cell lysate at 20 µgLane 4 : HEK-293 (human epithelial cell line from embryonic kidney) cell lysate at 20 µgLane 5 : Caco-2 (human colorectal adenocarcinoma cell line) cell lysate at 10 µgSecondaryAll lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilutionDeveloped using the ECL technique.Predicted band size: 59 kDaObserved band size: 25,53,65 kDa why is the actual band size different from the predicted?Exposure time : Lanes 1-4: 3 minutes; lane 5: 1 minute.Blocking/Dilution buffer: 5% NFDM/TBST.The molecular weight observed is consistent with what has been described in the literature (PMID: 18647175, PMID: 19821483). All lanes : Anti-Occludin antibody [EPR20992] (ab216327) at 1/1000 dilutionLane 1 : Rat brain lysateLane 2 : Mouse brain lysateLane 3 : Mouse uterus lysateLysates/proteins at 10 µg per lane.SecondaryAll lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilutionDeveloped using the ECL technique.Predicted band size: 59 kDaObserved band size: 23,53,65 kDa why is the actual band size different from the predicted?Exposure time: 3 minutesBlocking/Dilution buffer: 5% NFDM/TBST.The molecular weight observed is consistent with what has been described in the literature (PMID: 18647175, PMID: 19821483). Immunohistochemical analysis of paraffin-embedded mouse kidney tissue labeling Occludin with ab216327 at 1/200 dilution, followed by Goat Anti-Rabbit IgG H L (HRP) ready to use. Cytoplasmic staining on distal tubules of mouse kidney is observed. Counter stained with Hematoxylin.Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H L (HRP) ready to use.Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. Immunohistochemical analysis of paraffin-embeddedrat kidney tissue labeling Occludin with ab216327 at 1/200 dilution, followed by Goat Anti-Rabbit IgG H L (HRP) ready to use. Cytoplasmic staining on distal tubules of rat kidney is observed. Counter stained with Hematoxylin.Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H L (HRP)ready to use.Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. Immunohistochemical analysis of paraffin-embedded human breast tissue labeling Occludin with ab216327 at 1/200 dilution, followed by Goat Anti-Rabbit IgG H L (HRP) ready to use. Membranous staining on human breast is observed. Counter stained with Hematoxylin.Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H L (HRP) ready to use.Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilizedMDCK (canine kidney cell line) cells labelingOccludin with ab216327 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H L (Alexa Fluor 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing membrane staining on MDCK (NBL-2) cell line.The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor 594) (ab195889) at 1/200 dilution (red).Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H L (Alexa Fluor 488) (ab150077) secondary antibody at 1/1000 dilution. Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized Caco-2 (human colorectal adenocarcinoma cell line) cell line labeling Occludin with ab216327 at 1/60 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H L (Alexa Fluor 488) (ab150077) at 1/2000 dilution was used as the secondary antibody. Occludinwas immunoprecipitated from 0.35 mg of Caco-2 (human colorectal adenocarcinoma cell line) whole cell lysate with ab216327 at 1/40 dilution.Western blot was performed from the immunoprecipitate using ab216327 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilutionLane 1:Caco-2 whole cell lysate 10 g (Input).Lane 2: ab216327 IP inCaco-2 whole cell lysate.Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab216327 inCaco-2 whole cell lysate.Blocking and dilution buffer and concentration: 5% NFDM/TBST.Exposure time: 1 second.The molecular weight observed is consistent with what has been described in the literature (PMID: 18647175, PMID: 19821483). To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on. Click here to view the general protocols To download a Certificate of Compliance, please enter your Lot number below:Lot number 发表研究结果有使用 ab216327？请让我们知道，以便我们可以引用本数据表中的参考文章。 ab216327 被引用在 46 文献中. Shi Y et al. Low molecular weight heparin (nadroparin) improves placental permeability in rats with gestational diabetes mellitus via reduction of tight junction factors. Mol Med Rep 21:623-630 (2020).PubMed: 31974593 Yuan Y et al. Salidroside ameliorates liver metabonomics in relation to modified gut-liver FXR signaling in furan-induced mice. Food Chem Toxicol 140:111311 (2020).PubMed: 32251705 Li Y et al. Protective effect of emodin on intestinal epithelial tight junction barrier integrity in rats with sepsis induced by cecal ligation and puncture. Exp Ther Med 19:3521-3530 (2020).PubMed: 32346413 Liu X et al. Determination of the available energy values and amino acid digestibility of Flammulina velutipes stem waste and its effects on carcass trait and meat quality fed to growing-finishing pigs. J Anim Sci Biotechnol 11:41 (2020).PubMed: 32391146 Zheng J et al. The CXCR4/miR-125b/FoxP3 axis regulates the function of the epithelial barrier via autophagy in allergic rhinitis. Am J Transl Res 12:2570-2584 (2020).PubMed: 32655791 View all Publications for this product Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) Concentration: 2% Temperature: RT C Blocking step BSA as blocking agent for 30 minute(s) Concentration: 3% Temperature: 20 C Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) Concentration: 5% Temperature: 25 C Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) Concentration: 5% Temperature: 25 CPlease note: All products are FOR RESEARCH USE ONLY. 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