The Mouse Phosphatidylinositol-3,4,5-trisphosphate 5-Phosphatase 1 (INPP5D) ELISA Kit is specifically designed for the accurate detection of INPP5D levels in mouse serum, plasma, and cell culture supernatants. This kit offers high sensitivity and specificity for reliable and reproducible results, making it suitable for a variety of research applications.INPP5D is an important enzyme involved in the regulation of phosphoinositide signaling pathways, playing a key role in various cellular processes such as cell growth, survival, and differentiation.
Dysregulation of INPP5D has been implicated in cancer, immune disorders, and inflammatory diseases, highlighting its significance as a biomarker for studying these conditions and potentially developing targeted therapies.Overall, the Mouse Phosphatidylinositol-3,4,5-trisphosphate 5-Phosphatase 1 (INPP5D) ELISA Kit provides a valuable tool for researchers looking to investigate the role of INPP5D in various biological processes and diseases in mouse models.
Phosphatidylinositol (PtdIns) phosphatase that specifically hydrolyzes the 5-phosphate of phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) to produce PtdIns(3,4)P2, thereby negatively regulating the PI3K (phosphoinositide 3-kinase) pathways (By similarity). Able also to hydrolyzes the 5-phosphate of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P3) and inositol 1,3,4,5-tetrakisphosphate (PubMed:9367159). Acts as a negative regulator of B-cell antigen receptor signaling. Mediates signaling from the FC-gamma-RIIB receptor (FCGR2B), playing a central role in terminating signal transduction from activating immune/hematopoietic cell receptor systems. Acts as a negative regulator of myeloid cell proliferation/survival and chemotaxis, mast cell degranulation, immune cells homeostasis, integrin alpha-IIb/beta-3 signaling in platelets and JNK signaling in B-cells. Regulates proliferation of osteoclast precursors, macrophage programming, phagocytosis and activation and is required for endotoxin tolerance. Involved in the control of cell-cell junctions, CD32a signaling in neutrophils and modulation of EGF-induced phospholipase C activity. Key regulator of neutrophil migration, by governing the formation of the leading edge and polarization required for chemotaxis. Modulates FCGR3/CD16-mediated cytotoxicity in NK cells. Mediates the activin/TGF-beta-induced apoptosis through its Smad-dependent expression.
Uniprot:
Q9ES52
Sample Type:
Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:
Natural and recombinant mouse Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1
Sub Unit:
Interacts with tyrosine phosphorylated form of SHC1 (PubMed:8654924, PubMed:8643691, PubMed:10068665, PubMed:9083021, PubMed:9099679, PubMed:10395202). Interacts with tyrosine phosphorylated form of DOK1 (PubMed:11031258). Interacts with tyrosine phosphorylated form of DOK3 (PubMed:14993273). Interacts with tyrosine phosphorylated form of SLAMF1/CD150 (By similarity). Interacts with PTPN11/SHP-2 in response to IL-3 (PubMed:9110989, PubMed:9393882). Interacts with receptor EPOR (PubMed:10660611). Interacts with receptors MS4A2/FCER1B and FCER1G (By similarity). Interacts with receptors FCGR2B and FCGR3 (PubMed:10395202, PubMed:11016922, PubMed:10779347, PubMed:15456754, PubMed:12393695). Interacts with receptor FCGR2A, leading to regulate gene expression during the phagocytic process (PubMed:12370370). Interacts with GRB2 (PubMed:8643691, PubMed:10068665). Interacts with PLCG1 (PubMed:16000869). Interacts with tyrosine kinases SRC and TEC (PubMed:10794720, PubMed:15492005). Interacts with CRKL (PubMed:11031258). Interacts with c-Met/MET (PubMed:11896575). Interacts with MILR1 (tyrosine-phosphorylated) (PubMed:20526344). Isoform 5 interacts with IL6ST/gp130 (PubMed:17105399). Can weakly interact (via NPXY motif 2) with DAB2 (via PID domain); the interaction is impaired by tyrosine phosphorylation of the NPXY motif (PubMed:11247302).
Research Area:
Cancer
Subcellular Location:
Isoform 5 Cell membrane Peripheral membrane protein Constitutively present at the cell membrane (PubMed:11567986).
Storage:
Please see kit components below for exact storage details
Note:
For research use only
UniProt Protein Function:
SHIP: an SH2-containing inositol phosphatase. A hemopoietic-specific phosphatase that regulates cell survival, growth, cell cycle arrest and apoptosis. Hydrolyzes Ins(1,3,4,5)P4 and PtdIns(3,4,5)P3. A cytosolic protein with a SH2 domain in its N-terminus and two NPXY Shc binding motifs at its C-terminus. Upon receptor cross-linking, SHIP is first recruited to the plasma membrane by its SH2, followed by tyrosine phosphorylation on the NPXY motif. Both membrane relocalization and phosphorylation are essential for its regulatory function. Associates with SHC after cytokine stimulation.Protein type: EC 3.1.3.86; Phosphatase, lipid; Motility/polarity/chemotaxisCellular Component: cortical cytoskeleton; membrane; cytoplasm; plasma membrane; actin filament; intracellular; cytosolMolecular Function: protein binding; hydrolase activity; PTB domain binding; inositol-4,5-bisphosphate 5-phosphatase activity; inositol-polyphosphate 5-phosphatase activity; SH3 domain bindingBiological Process: negative regulation of osteoclast differentiation; positive regulation of apoptosis; apoptosis; immune system process; positive regulation of erythrocyte differentiation; negative regulation of immune response; negative regulation of B cell activation; negative regulation of interleukin-6 biosynthetic process; negative regulation of B cell proliferation; determination of adult life span; negative regulation of monocyte differentiation; negative regulation of neutrophil differentiation; negative regulation of granulocyte differentiation; negative regulation of signal transduction; positive regulation of lymphocyte differentiation; negative regulation of cell proliferation; dephosphorylation; phosphoinositide dephosphorylation; positive regulation of B cell differentiation; immunoglobulin mediated immune response; negative regulation of bone resorption
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.
