Rat Pdgfra ELISA Kit (RTEB0087)- High Sensitivity

Product Name:	Rat Alpha-type platelet-derived growth factor receptor (Pdgfra) ELISA Kit
SKU:	RTEB0087
Size:	96T
Target:	Rat Alpha-type platelet-derived growth factor receptor (Pdgfra)
Synonyms:	Alpha platelet-derived growth factor receptor, Alpha-type platelet-derived growth factor receptor, CD140 antigen-like family member A, Platelet-derived growth factor alpha receptor, CD140a, PDGF-R-alpha
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Rat
Detection Range:	0.156-10ng/mL
Sensitivity:	0.078ng/mL

Intra CV:	Provided with the Kit
Inter CV:	Provided with the Kit
Linearity:	Provided with the Kit
Recovery:	Provided with the Kit
Function:	Tyrosine-protein kinase that acts as a cell-surface receptor for PDGFA, PDGFB and PDGFC and plays an essential role in the regulation of embryonic development, cell proliferation, survival and chemotaxis. Depending on the context, promotes or inhibits cell proliferation and cell migration. Plays an important role in the differentiation of bone marrow-derived mesenchymal stem cells. Required for normal skeleton development and cephalic closure during embryonic development. Required for normal development of the mucosa lining the gastrointestinal tract, and for recruitment of mesenchymal cells and normal development of intestinal villi. Plays a role in cell migration and chemotaxis in wound healing. Plays a role in platelet activation, secretion of agonists from platelet granules, and in thrombin-induced platelet aggregation. Binding of its cognate ligands - homodimeric PDGFA, homodimeric PDGFB, heterodimers formed by PDGFA and PDGFB or homodimeric PDGFC -leads to the activation of several signaling cascades; the response depends on the nature of the bound ligand and is modulated by the formation of heterodimers between PDGFRA and PDGFRB. Phosphorylates PIK3R1, PLCG1, and PTPN11. Activation of PLCG1 leads to the production of the cellular signaling molecules diacylglycerol and inositol 1,4,5-trisphosphate, mobilization of cytosolic Ca(2+) and the activation of protein kinase C. Phosphorylates PIK3R1, the regulatory subunit of phosphatidylinositol 3-kinase, and thereby mediates activation of the AKT1 signaling pathway. Mediates activation of HRAS and of the MAP kinases MAPK1/ERK2 and/or MAPK3/ERK1. Promotes activation of STAT family members STAT1, STAT3 and STAT5A and/or STAT5B. Receptor signaling is down-regulated by protein phosphatases that dephosphorylate the receptor and its down-stream effectors, and by rapid internalization of the activated receptor.

Uniprot:	P20786
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant rat Platelet-derived growth factor receptor alpha
Sub Unit:	Interacts with homodimeric PDGFA, PDGFB and PDGFC, and with heterodimers formed by PDGFA and PDGFB. Monomer in the absence of bound ligand. Interaction with dimeric PDGFA, PDGFB and/or PDGFC leads to receptor dimerization, where both PDGFRA homodimers and heterodimers with PDGFRB are observed. Interacts (tyrosine phosphorylated) with SHB (via SH2 domain). Interacts (tyrosine phosphorylated) with SHF (via SH2 domain). Interacts (tyrosine phosphorylated) with SRC (via SH2 domain). Interacts (tyrosine phosphorylated) with PIK3R1. Interacts (tyrosine phosphorylated) with PLCG1 (via SH2 domain). Interacts (tyrosine phosphorylated) with CRK, GRB2 and GRB7.
Research Area:	Cancer
Subcellular Location:	Cell membrane Single-pass type I membrane protein The activated receptor is rapidly internalized and degraded.
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	PDGFRA: a receptor tyrosine kinase of the PDGFR family that binds members of the platelet-derived growth factor family. The identity of the growth factor bound determines whether the functional receptor is a homodimer or a heterodimer, composed of both PDGFR-alpha and -beta. Ligand binding induces receptor dimerization and autophosphorylation. Particularly important for kidney development since mice heterozygous for the receptor exhibit defective kidney phenotypes. Chromosomal rearrangments activate PDGFRalpha by fusion to BCR, causing atypical chronic myelogenous leukemia (CML), and to FIP1L1, causing idiopathic hypereosinophilic syndrome. Activating point mutations cause a minority of gastrointestinal stromal tumors (GIST). Promoter polymorphisms linked to neural tube defects including spina bifida, verified by mouse mutant model. Inhibitors: Gleevec, Sutent. OMIM: Two alternatively-spliced isoforms have been described.Protein type: Oncoprotein; Membrane protein, integral; Protein kinase, tyrosine (receptor); Protein kinase, TK; EC 2.7.10.1; Kinase, protein; TK group; PDGFR familyChromosomal Location of Human Ortholog: 4q12Cellular Component: microvillus; membrane; integral to plasma membrane; cytoplasm; plasma membrane; nucleus; intrinsic to plasma membrane; external side of plasma membraneMolecular Function: vascular endothelial growth factor receptor activity; protein binding; protein homodimerization activity; platelet-derived growth factor binding; platelet-derived growth factor receptor binding; platelet-derived growth factor alpha-receptor activity; transmembrane receptor protein tyrosine kinase activity; ATP bindingBiological Process: estrogen metabolic process; extracellular matrix organization and biogenesis; regulation of chemotaxis; peptidyl-tyrosine phosphorylation; nerve growth factor receptor signaling pathway; wound healing; viral reproduction; protein amino acid autophosphorylation; platelet-derived growth factor receptor signaling pathway; cardiac myofibril assembly; palate development; positive regulation of fibroblast proliferation; elevation of cytosolic calcium ion concentration; Leydig cell differentiation; embryonic digestive tract morphogenesis; luteinization; positive regulation of cell proliferation; male genitalia development; epidermal growth factor receptor signaling pathway; fibroblast growth factor receptor signaling pathway; phosphoinositide-mediated signaling; adrenal gland development; in utero embryonic development; positive regulation of phosphoinositide 3-kinase activity; embryonic cranial skeleton morphogenesis; odontogenesis of dentine-containing teeth; embryonic skeletal morphogenesis; positive regulation of phosphoinositide 3-kinase cascade; cell activation; innate immune response; hemopoietic progenitor cell differentiation; positive regulation of DNA replication; lung development; positive regulation of cell migrationDisease: Gastrointestinal Stromal Tumor; Hypereosinophilic Syndrome, Idiopathic
UniProt Protein Details:
NCBI Summary:	This gene encodes a cell surface tyrosine kinase receptor for members of the platelet-derived growth factor family. These growth factors are mitogens for cells of mesenchymal origin. The identity of the growth factor bound to a receptor monomer determines whether the functional receptor is a homodimer or a heterodimer, composed of both platelet-derived growth factor receptor alpha and beta polypeptides. Studies suggest that this gene plays a role in organ development, wound healing, and tumor progression. Mutations in this gene have been associated with idiopathic hypereosinophilic syndrome, somatic and familial gastrointestinal stromal tumors, and a variety of other cancers. [provided by RefSeq, Mar 2012]
UniProt Code:	P20786
NCBI GenInfo Identifier:	5453870
NCBI Gene ID:	5156
NCBI Accession:	NP_006197.1
UniProt Secondary Accession:	P20786,Q6P4H5, Q96KZ7, Q9UD28, B2RE69, E9PBH0
UniProt Related Accession:	P20786,P16234
Molecular Weight:
NCBI Full Name:	platelet-derived growth factor receptor alpha
NCBI Synonym Full Names:	platelet-derived growth factor receptor, alpha polypeptide
NCBI Official Symbol:	PDGFRA
NCBI Official Synonym Symbols:	CD140A; PDGFR2; PDGFR-2; RHEPDGFRA
NCBI Protein Information:	platelet-derived growth factor receptor alpha; PDGF-R-alpha; CD140a antigen; PDGFRA/BCR fusion; CD140 antigen-like family member A; platelet-derived growth factor receptor 2; alpha-type platelet-derived growth factor receptor; rearranged-in-hypereosinophilia-platelet derived growth factor receptor alpha fusion protein
UniProt Protein Name:	Platelet-derived growth factor receptor alpha
UniProt Synonym Protein Names:	Alpha platelet-derived growth factor receptor; Alpha-type platelet-derived growth factor receptor; CD140 antigen-like family member A; CD140a antigen; Platelet-derived growth factor alpha receptor; Platelet-derived growth factor receptor 2; PDGFR-2; CD_antigen: CD140a
Protein Family:	Platelet-derived growth factor receptor
UniProt Gene Name:	PDGFRA
UniProt Entry Name:	PGFRA_HUMAN

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.