Product Name:	Human TJP1(Tight junction protein ZO-1) ELISA Kit
Product Code:	HUFI03026
Size:	96 Assays
Alias:	Tight junction protein 1, Tight junction protein ZO 1, TJP1, ZO 1, ZO1, ZO-1, Zona occludens protein 1, Zonula occludens protein 1
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human TJP1 concentrations in serum plasma and other biological fluids.
Sensitivity:	0.188ng/ml
Range:	0.313-20ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:	Matrices listed below were spiked with certain level of Human TJP1 and the recovery rates were calculated by comparing the measured value to the expected amount of Human TJP1 in samples. Enquire for more information.
Linearity:	The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human TJP1 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected. Enquire for more information.
CV(%):	Intra-Assay: CV<8% Inter-Assay: CV<10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

Uniprot

Q07157

UniProt Protein Function:	ZO1: a scaffolding protein of the MAGUK (membrane-associated guanylate kinase) family associated with tight junctions (TJ). May mediate dynamic changes in the composition of TJs. The N-terminal may be involved in transducing a signal required for tight junction assembly, while the C-terminal may have specific properties of tight junctions. The alpha domain might be involved in stabilizing junctions. Plays a role in the regulation of cell migration by targeting the protein kinase MRCKb to the leading edge of migrating cells. Interacts with BVES (via the C-terminus cytoplasmic tail). Interacts with HSPA4 and KIRREL1. Forms homodimers and heterodimers with ZO-2 or ZO-3. Interacts with occludin, CALM, claudins, CGN, CXADR, GJA12, GJD3 and UBN1. Interacts (via ZU5 domain) with MRCKb and MYZAP. Interacts (via PDZ domain) with Cx43. ZO-2 and ZO-3 shuttle between the TJ and the nucleus, where they may regulate gene expression. TJ, or zonula occludens, are the closely associated areas of two cells whose membranes are tightly joined together. They are composed of a branching network of strands, the efficiency of which increases exponentially with the number of strands. The strands associate with transmembrane proteins that bind to similar proteins on adjacent cells, and with submembranous proteins that are anchored to the actin component of the cytoskeleton. Thus, TJs join together the cytoskeletons of adjacent cells. They endow tissues with substantial form, shape and location, enforce cellular polarity, and form barriers to molecules and pathogens. Molecules forming the membranous part of TJs include occludin, claudins, tricellulin and junctional adhesion molecules. These molecules interact with the proteins ZO-1, ZO-2 and ZO-3. Tight junction proteins can be up- or downregulated in cancer and are involved in the epithelial-mesenchymal transitions (EMT) in tumors. Alternative splicing produces two isoforms. The long isoform is found in most epithelial cell junctions. The short isoform is found both in endothelial cells and the highly specialized epithelial junctions of renal glomeruli and Sertoli cells of the seminiferous tubules.
UniProt Protein Details:	Protein type:Motility/polarity/chemotaxis; Adaptor/scaffold Chromosomal Location of Human Ortholog: 15q13 Cellular Component: apical junction complex; intercellular canaliculus; tight junction; apicolateral plasma membrane; basolateral plasma membrane; cytosol; cell-cell adherens junction; apical part of cell; cytoplasm; apical plasma membrane; plasma membrane; gap junction; nucleus; cell junction Molecular Function:protein C-terminus binding; calmodulin binding; protein domain specific binding; protein binding Biological Process: response to drug; intercellular junction assembly; response to ethanol; sensory perception of sound; apoptosis; negative regulation of vascular permeability; blastocyst formation; response to lipopolysaccharide; cell structure disassembly during apoptosis
NCBI Summary:	This gene encodes a protein located on a cytoplasmic membrane surface of intercellular tight junctions. The encoded protein may be involved in signal transduction at cell-cell junctions. Alternative splicing of this gene results in multiple transcript variants. [provided by RefSeq, Jul 2014]
UniProt Code:	Q07157
NCBI GenInfo Identifier:	85700443
NCBI Gene ID:	7082
NCBI Accession:	Q07157.3
UniProt Secondary Accession:	Q07157,Q2NKP3, Q4ZGJ6, B4E3K1,
UniProt Related Accession:	Q07157
Molecular Weight:	1748
NCBI Full Name:	Tight junction protein ZO-1
NCBI Synonym Full Names:	tight junction protein 1
NCBI Official Symbol:	TJP1
NCBI Official Synonym Symbols:	ZO-1
NCBI Protein Information:	tight junction protein ZO-1; zona occludens 1; zonula occludens 1 protein
UniProt Protein Name:	Tight junction protein ZO-1
UniProt Synonym Protein Names:	Tight junction protein 1; Zona occludens protein 1; Zonula occludens protein 1
UniProt Gene Name:	TJP1
UniProt Entry Name:	ZO1_HUMAN

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.