Product Name:	Human Ectonucleotide pyrophosphatase / phosphodiesterase family member 1 / ENPP1 ELISA Kit
Product Code:	HUFI00899
Size:	96 Assays
Alias:	ENPP1, Ly-41 Antigen, M6S1, NPP1, NPPS, PCA1, PDNP1, E-NPP 1, PC1, PC-1, ARHR2, Membrane component chromosome 6 surface marker 1, alkaline phosphodiesterase 1, plasma-cell membrane glycoprotein 1
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human ENPP1 concentrations in serum plasma and other biological fluids.
Sensitivity:	0.094ng/ml
Range:	0.156-10ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human ENPP1 and the recovery rates were calculated by comparing the measured value to the expected amount of Human ENPP1 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	85-103	93
EDTA plasma(n=5)	87-105	97
UFH plasma(n=5)	86-105	98

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human ENPP1 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)	90-100%	92-102%	92-104%
EDTA plasma(n=5)	84-100%	84-92%	82-96%
UFH plasma(n=5)	85-99%	84-100%	83-100%

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

P22413

UniProt Protein Function:	ENPP1: Involved primarily in ATP hydrolysis at the plasma membrane. Plays a role in regulating pyrophosphate levels, and functions in bone mineralization and soft tissue calcification. In vitro, has a broad specificity, hydrolyzing other nucleoside 5' triphosphates such as GTP, CTP, TTP and UTP to their corresponding monophosphates with release of pyrophosphate and diadenosine polyphosphates, and also 3',5'-cAMP to AMP. May also be involved in the regulation of the availability of nucleotide sugars in the endoplasmic reticulum and Golgi, and the regulation of purinergic signaling. Appears to modulate insulin sensitivity. Homodimer; disulfide-linked. Interacts with INSR. Expressed in plasma cells and also in a number of non-lymphoid tissues, including the distal convoluted tubule of the kidney, chondrocytes and epididymis. At low concentrations of ATP, a phosphorylated intermediate is formed which inhibits further hydrolysis. Belongs to the nucleotide pyrophosphatase/phosphodiesterase family.
UniProt Protein Details:	Protein type:Membrane protein, integral; Cofactor and Vitamin Metabolism - nicotinate and nicotinamide; Nucleotide Metabolism - purine; Cofactor and Vitamin Metabolism - pantothenate and CoA biosynthesis; EC 3.1.4.1; Phosphatase (non-protein); Motility/polarity/chemotaxis; Carbohydrate Metabolism - starch and sucrose; Cofactor and Vitamin Metabolism - riboflavin; Phosphodiesterase; EC 3.6.1.9 Chromosomal Location of Human Ortholog: 6q22-q23 Cellular Component: extracellular space; cell surface; lysosomal membrane; integral to plasma membrane; basolateral plasma membrane; integral to membrane; plasma membrane Molecular Function:phosphodiesterase I activity; nucleotide diphosphatase activity; protein homodimerization activity; 3'-phosphoadenosine 5'-phosphosulfate binding; zinc ion binding; calcium ion binding; nucleoside-triphosphate diphosphatase activity; insulin receptor binding; polysaccharide binding; protein binding; nucleic acid binding; scavenger receptor activity; ATP binding Biological Process: receptor-mediated endocytosis; generation of precursor metabolites and energy; sequestering of triacylglycerol; vitamin metabolic process; nucleoside triphosphate catabolic process; negative regulation of insulin receptor signaling pathway; bone remodeling; negative regulation of fat cell differentiation; phosphate metabolic process; 3'-phosphoadenosine 5'-phosphosulfate metabolic process; riboflavin metabolic process; negative regulation of glucose import; cellular phosphate ion homeostasis; cellular response to insulin stimulus; biomineral formation; negative regulation of ossification; immune response; negative regulation of protein amino acid autophosphorylation; negative regulation of cell growth; regulation of bone mineralization; water-soluble vitamin metabolic process; inorganic diphosphate transport; negative regulation of glycogen biosynthetic process Disease: Obesity; Cole Disease; Arterial Calcification, Generalized, Of Infancy, 1; Hypophosphatemic Rickets, Autosomal Recessive, 2; Diabetes Mellitus, Noninsulin-dependent
NCBI Summary:	This gene is a member of the ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP) family. The encoded protein is a type II transmembrane glycoprotein comprising two identical disulfide-bonded subunits. This protein has broad specificity and cleaves a variety of substrates, including phosphodiester bonds of nucleotides and nucleotide sugars and pyrophosphate bonds of nucleotides and nucleotide sugars. This protein may function to hydrolyze nucleoside 5' triphosphates to their corresponding monophosphates and may also hydrolyze diadenosine polyphosphates. Mutations in this gene have been associated with 'idiopathic' infantile arterial calcification, ossification of the posterior longitudinal ligament of the spine (OPLL), and insulin resistance. [provided by RefSeq, Jul 2008]
UniProt Code:	P22413
NCBI GenInfo Identifier:	23503088
NCBI Gene ID:	5167
NCBI Accession:	P22413.2
UniProt Related Accession:	P22413
Molecular Weight:
NCBI Full Name:	Ectonucleotide pyrophosphatase/phosphodiesterase family member 1
NCBI Synonym Full Names:	ectonucleotide pyrophosphatase/phosphodiesterase 1
NCBI Official Symbol:	ENPP1
NCBI Official Synonym Symbols:	M6S1; NPP1; NPPS; PC-1; PCA1; ARHR2; COLED; PDNP1
NCBI Protein Information:	ectonucleotide pyrophosphatase/phosphodiesterase family member 1
UniProt Protein Name:	Ectonucleotide pyrophosphatase/phosphodiesterase family member 1
UniProt Synonym Protein Names:	Membrane component chromosome 6 surface marker 1; Phosphodiesterase I/nucleotide pyrophosphatase 1; Plasma-cell membrane glycoprotein PC-1Including the following 2 domains:Alkaline phosphodiesterase I (EC:3.1.4.1); Nucleotide pyrophosphatase (EC:3.6.1.9); NPPase
Protein Family:	Ectonucleotide pyrophosphatase/phosphodiesterase family
UniProt Gene Name:	ENPP1
UniProt Entry Name:	ENPP1_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.