Mouse Ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (Enpp1) ELISA Kit
The Mouse Ectonucleotide Pyrophosphatase/Phosphodiesterase Family Member 1 (ENPP1) ELISA Kit is specifically designed for the quantitative detection of ENPP1 levels in mouse serum, plasma, and tissue lysates. This kit offers exceptional sensitivity and specificity, ensuring accurate and consistent results for various research applications.ENPP1 is a key enzyme involved in the regulation of phosphate metabolism, bone mineralization, and insulin sensitivity. Dysregulation of ENPP1 has been associated with conditions such as insulin resistance, diabetes, and vascular calcification, highlighting its importance as a potential biomarker for studying these diseases and exploring therapeutic interventions.
With the Mouse ENPP1 ELISA Kit, researchers can reliably measure ENPP1 levels in mouse samples, providing valuable insights into the role of this enzyme in health and disease. Its user-friendly format and robust performance make it a valuable tool for advancing research in the field of metabolic disorders and skeletal diseases.
Product Name:
Mouse Ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (Enpp1) ELISA Kit
SKU:
MOEB0755
Size:
96T
Target:
Mouse Ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (Enpp1)
Appears to modulate insulin sensitivity (By similarity). By generating PPi, plays a role in regulating pyrophosphate levels, and functions in bone mineralization and soft tissue calcification. PPi inhibits mineralization by binding to nascent hydroxyapatite (HA) crystals, thereby preventing further growth of these crystals. Preferentially hydrolyzes ATP, but can also hydrolyze 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.
Uniprot:
P06802
Sample Type:
Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:
Natural and recombinant mouse Ectonucleotide pyrophosphatase/phosphodiesterase family member 1
Sub Unit:
Homodimer. The secreted form is monomeric. Interacts with INSR.
Research Area:
Metabolism
Subcellular Location:
Cell membrane Single-pass type II membrane protein Basolateral cell membrane Single-pass type II membrane protein Secreted Targeted to the basolateral membrane in polarized epithelial cells and in hepatocytes, and to matrix vesicles in osteoblasts. In bile duct cells and cancer cells, located to the apical cytoplasmic side (By similarity). The proteolytically processed form is secreted.
Storage:
Please see kit components below for exact storage details
Note:
For research use only
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.Protein type: EC 3.6.1.9; Phosphatase (non-protein); Cofactor and Vitamin Metabolism - riboflavin; Phosphodiesterase; Motility/polarity/chemotaxis; Membrane protein, integral; Nucleotide Metabolism - purine; EC 3.1.4.1; Cofactor and Vitamin Metabolism - nicotinate and nicotinamide; Cofactor and Vitamin Metabolism - pantothenate and CoA biosynthesis; Carbohydrate Metabolism - starch and sucroseCellular Component: extracellular space; cell surface; membrane; integral to plasma membrane; lysosomal membrane; integral to membrane; plasma membrane; extracellular regionMolecular Function: nucleotide diphosphatase activity; phosphodiesterase I activity; protein homodimerization activity; zinc ion binding; hydrolase activity; metal ion binding; calcium ion binding; nucleoside-triphosphate diphosphatase activity; insulin receptor binding; polysaccharide binding; nucleic acid binding; catalytic activity; ATP binding; scavenger receptor activityBiological Process: generation of precursor metabolites and energy; sequestering of triacylglycerol; 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; 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; inorganic diphosphate transport; negative regulation of glycogen biosynthetic process
UniProt Protein Details:
NCBI Summary:
This gene encodes a member of the nucleoside pyrophosphatase/phosphodiesterase family of enzymes that catalyzes the hydrolysis of pyrophosphate and phosphodiester bonds in nucleotide triphosphates and oligonucleotides, respectively, to generate nucleoside 5'-monophosphates. The encoded protein is a type II transmembrane glycoprotein that negatively regulates bone mineralization. Mice harboring a nonsense mutation in this gene, termed tiptoe walking (ttw), exhibit ectopic ossification of the spinal ligaments. The encoded protein binds to the insulin receptor, inhibits downstream signaling events and induces insulin resistance and glucose tolerance. This gene is located adjacent to a paralog on chromosome 10. Alternative splicing results in multiple transcript variants encoding different isoforms. [provided by RefSeq, Apr 2015]
Ectonucleotide pyrophosphatase/phosphodiesterase family
UniProt Gene Name:
Enpp1
UniProt Entry Name:
ENPP1_MOUSE
Component
Quantity (96 Assays)
Storage
ELISA Microplate (Dismountable)
8×12 strips
-20°C
Lyophilized Standard
2
-20°C
Sample Diluent
20ml
-20°C
Assay Diluent A
10mL
-20°C
Assay Diluent B
10mL
-20°C
Detection Reagent A
120µL
-20°C
Detection Reagent B
120µL
-20°C
Wash Buffer
30mL
4°C
Substrate
10mL
4°C
Stop Solution
10mL
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
*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.
Step
1.
Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.
Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.
Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.
Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.
Repeat the wash process for five times as conducted in step 3.
6.
Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.
Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.
Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.
After experiment, store all reagents according to the specified storage temperature respectively until their expiry.
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.
