Product Name:	Human Heparanase / HPSE ELISA Kit
Product Code:	HUFI00775
Size:	96 Assays
Alias:	HPSE, Heparanase, HEP, HPA, HPA1, HPR1, HPSE1, HSE1, Endo-glucoronidase, Heparanase-1
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human HPSE concentrations in serum plasma and other biological fluids.
Sensitivity:	46.875pg/ml
Range:	78.125-5000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human HPSE and the recovery rates were calculated by comparing the measured value to the expected amount of Human HPSE in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	91-98	94
EDTA plasma(n=5)	87-95	91
UFH plasma(n=5)	85-104	93

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human HPSE and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	86-102%	91-104%	95-101%
EDTA plasma(n=5)	83-100%	83-98%	84-100%
UFH plasma(n=5)	80-98%	87-98%	84-100%

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

Q9Y251

UniProt Protein Function:	HPSE: Endoglycosidase that cleaves heparan sulfate proteoglycans (HSPGs) into heparan sulfate side chains and core proteoglycans. Participates in extracellular matrix (ECM) degradation and remodeling. Selectively cleaves the linkage between a glucuronic acid unit and an N-sulfo glucosamine unit carrying either a 3-O-sulfo or a 6-O-sulfo group. Can also cleave the linkage between a glucuronic acid unit and an N-sulfo glucosamine unit carrying a 2-O-sulfo group, but not linkages between a glucuronic acid unit and a 2-O-sulfated iduronic acid moiety. It is essentially inactive at neutral pH but becomes active under acidic conditions such as during tumor invasion and in inflammatory processes. Facilitates cell migration associated with metastasis, wound healing and inflammation. Enhances shedding of syndecans, and increases endothelial invasion and angiogenesis in myelomas. Acts as procoagulant by increasing the generation of activation factor X in the presence of tissue factor and activation factor VII. Increases cell adhesion to the extacellular matrix (ECM), independent of its enzymatic activity. Induces AKT1/PKB phosphorylation via lipid rafts increasing cell mobility and invasion. Heparin increases this AKT1/PKB activation. Regulates osteogenesis. Enhances angiogenesis through up- regulation of SRC-mediated activation of VEGF. Implicated in hair follicle inner root sheath differentiation and hair homeostasis. Belongs to the glycosyl hydrolase 79 family.
UniProt Protein Details:	Protein type:EC 3.2.1.166; Glycan Metabolism - glycosaminoglycan degradation; Hydrolase; Extracellular matrix; Secreted, signal peptide; Secreted Chromosomal Location of Human Ortholog: 4q21.3 Cellular Component: nucleoplasm; lysosomal lumen; intracellular membrane-bound organelle; lysosomal membrane; lysosome; extracellular region; nucleus; lipid raft Molecular Function:protein dimerization activity; syndecan binding; protein binding; beta-glucuronidase activity; heparanase activity Biological Process: positive regulation of hair follicle development; proteoglycan metabolic process; positive regulation of protein kinase B signaling cascade; glycosaminoglycan catabolic process; heparan sulfate proteoglycan catabolic process; positive regulation of blood coagulation; glycosaminoglycan metabolic process; cell-matrix adhesion; positive regulation of osteoblast proliferation; carbohydrate metabolic process; pathogenesis; regulation of hair follicle development
NCBI Summary:	Heparan sulfate proteoglycans are major components of the basement membrane and extracellular matrix. The protein encoded by this gene is an enzyme that cleaves heparan sulfate proteoglycans to permit cell movement through remodeling of the extracellular matrix. In addition, this cleavage can release bioactive molecules from the extracellular matrix. Several transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Sep 2011]
UniProt Code:	Q9Y251
NCBI GenInfo Identifier:	296434532
NCBI Gene ID:	10855
NCBI Accession:	Q9Y251.2
UniProt Secondary Accession:	Q9Y251,Q53GE5, Q9UL39, A9JIG7, C7F7I3, C7F7I4, E9PCA9 E9PGR1,
UniProt Related Accession:	Q9Y251
Molecular Weight:	42,791 Da
NCBI Full Name:	Heparanase
NCBI Synonym Full Names:	heparanase
NCBI Official Symbol:	HPSE
NCBI Official Synonym Symbols:	HPA; HPA1; HPR1; HSE1; HPSE1
NCBI Protein Information:	heparanase; endo-glucoronidase; heparanase-1
UniProt Protein Name:	Heparanase
UniProt Synonym Protein Names:	Endo-glucoronidase; Heparanase-1; Hpa1
Protein Family:	HpaII very short patch repair endonuclease
UniProt Gene Name:	HPSE
UniProt Entry Name:	HPSE_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.