Rat Met ELISA Kit (RTEB1672)- High Sensitivity

Product Name:	Rat Hepatocyte growth factor receptor (Met) ELISA Kit
SKU:	RTEB1672
Size:	96T
Target:	Rat Hepatocyte growth factor receptor (Met)
Synonyms:	HGF/SF receptor, Proto-oncogene c-Met, Scatter factor receptor, Tyrosine-protein kinase Met, SF receptor, HGF receptor
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Rat
Detection Range:	78-5000pg/mL
Sensitivity:	40.4pg/mL

Intra CV:

5.3%

Inter CV:

9.1%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	85-97%	105-113%	103-113%	106-116%
EDTA Plasma(N=5)	96-105%	81-93%	105-114%	84-93%
Heparin Plasma(N=5)	99-109%	107-117%	100-112%	103-116%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	94	88-100
Plasma	96	90-102

Function:

Receptor tyrosine kinase that transduces signals from the extracellular matrix into the cytoplasm by binding to hepatocyte growth factor/HGF ligand. Regulates many physiological processes including proliferation, scattering, morphogenesis and survival. Ligand binding at the cell surface induces autophosphorylation of MET on its intracellular domain that provides docking sites for downstream signaling molecules. Following activation by ligand, interacts with the PI3-kinase subunit PIK3R1, PLCG1, SRC, GRB2, STAT3 or the adapter GAB1. Recruitment of these downstream effectors by MET leads to the activation of several signaling cascades including the RAS-ERK, PI3 kinase-AKT, or PLCgamma-PKC. The RAS-ERK activation is associated with the morphogenetic effects while PI3K/AKT coordinates prosurvival effects. During embryonic development, MET signaling plays a role in gastrulation, development and migration of muscles and neuronal precursors, angiogenesis and kidney formation. In adults, participates in wound healing as well as organ regeneration and tissue remodeling. Promotes also differentiation and proliferation of hematopoietic cells.

Uniprot:	P97523
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant rat Hepatocyte growth factor receptor
Sub Unit:	Heterodimer made of an alpha chain (50 kDa) and a beta chain (145 kDa) which are disulfide linked. Binds PLXNB1. Interacts when phosphorylated with downstream effectors including STAT3, PIK3R1, SRC, PCLG1, GRB2 and GAB1. Interacts with SPSB1, SPSB2 and SPSB4. Interacts with INPP5D/SHIP1. When phosphorylated at Tyr-1357, interacts with INPPL1/SHIP2. Interacts with RANBP9 and RANBP10, as well as SPSB1, SPSB2, SPSB3 and SPSB4. SPSB1 binding occurs in the presence and in the absence of HGF, however HGF treatment has a positive effect on this interaction. Interacts with MUC20; prevents interaction with GRB2 and suppresses hepatocyte growth factor-induced cell proliferation. Interacts with GRB10. Interacts with PTPN1 and PTPN2. Interacts with HSP90AA1 and HSP90AB1; the interaction suppresses MET kinase activity.
Subcellular Location:	Membrane Single-pass type I membrane protein
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	Met: a proto-oncogenic receptor tyrosine kinase with high affinity for hepatocyte growth factor. The primary single chain precursor protein is post-translationally cleaved to produce the 45 kDa alpha- and 145 kDa beta-subunits, which are disulfide linked to form the mature receptor. Ligand-binding induces autophosphorylation at multiple tyrosines, which recruit several downstream signaling components, including Gab1, c-Cbl and PI3 kinase. Activating point mutations cause hereditary papillary renal carcinoma. Mutations also seen in sporadic renal cell carcinoma and childhood hepatocellular carcinoma. Upregulation in carcinomas and sarcomas correlates with metastasis and poor outcome. Some gastric carcinomas harbor a translocation that creates an activated TPR-Met fusion protein. A small molecule inhibitor (PHA-665752) shows an effect in gastric carcinoma xenografts. Inhibitors: SU11274, PHA-665752, mAbs. Two alternatively spliced human isoforms have been reported.Protein type: EC 2.7.10.1; Protein kinase, tyrosine (receptor); Membrane protein, integral; Oncoprotein; Kinase, protein; Protein kinase, TK; TK group; Met familyCellular Component: extracellular space; neuron projection; cell surface; dendrite; postsynaptic density; integral to membrane; excitatory synapse; postsynaptic membrane; membrane; cell soma; cytoplasm; plasma membrane; basal plasma membraneMolecular Function: phospholipase binding; protein heterodimerization activity; protein-tyrosine kinase activity; protein complex binding; beta-catenin binding; phosphoinositide 3-kinase binding; hepatocyte growth factor receptor activity; protein phosphatase binding; ATP binding; protein kinase activityBiological Process: lactation; response to peptide hormone stimulus; muscle development; central nervous system neuron differentiation; peptidyl-tyrosine phosphorylation; activation of MAPK activity; myoblast proliferation; protein amino acid autophosphorylation; neuron migration; negative regulation of transcription from RNA polymerase II promoter; glucose homeostasis; response to organic cyclic substance; positive chemotaxis; pancreas development; response to wounding; endothelial cell morphogenesis; oligodendrocyte development; placenta development; response to drug; regulation of synaptic transmission; skeletal muscle development; positive regulation of mitosis; male gonad development; myotube differentiation; muscle cell migration; cell aging; hepatocyte growth factor receptor signaling pathway; positive regulation of dendrite morphogenesis; liver development; positive regulation of peptidyl-serine phosphorylation; sperm motility; branching morphogenesis of a tube; adult behavior; positive regulation of transcription from RNA polymerase II promoter; response to acidity; brain development; regulation of excitatory postsynaptic membrane potential; positive regulation of DNA replication
UniProt Protein Details:
NCBI Summary:	hepatocyte growth factor receptor; functions as a heterodimeric tyrosine kinase [RGD, Feb 2006]
UniProt Code:	P97523
NCBI GenInfo Identifier:	1771557
NCBI Gene ID:	24553
NCBI Accession:	CAA65582.1
UniProt Secondary Accession:	P97523
UniProt Related Accession:	P97523
Molecular Weight:	74.0 kDa
NCBI Full Name:	r.norvegicus mRNA for HGF receptor
NCBI Synonym Full Names:	MET proto-oncogene, receptor tyrosine kinase
NCBI Official Symbol:	Met
NCBI Official Synonym Symbols:	Hgfr
NCBI Protein Information:	hepatocyte growth factor receptor
UniProt Protein Name:
UniProt Synonym Protein Names:
Protein Family:	Metaxin
UniProt Gene Name:
UniProt Entry Name:

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.