Product Name:	Human Hepatocyte growth factor (HGF) ELISA Kit
Product Code:	HUEB0119
Alias:	Hepatocyte growth factor, Hepatopoietin-A, Scatter factor, SF, HGF, HPTA
Uniprot:	P14210
Reactivity:	Human
Range:	78-5000 pg/mL
Detection Method:	Sandwich
Size:	96 Assay
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	HGF: HGF is a potent mitogen for mature parenchymal hepatocyte cells, seems to be an hepatotrophic factor, and acts as growth factor for a broad spectrum of tissues and cell types. It has no detectable protease activity. Activating ligand for the receptor tyrosine kinase MET by binding and promoting its dimerization. Defects in HGF are the cause of deafness autosomal recessive type 39 (DFNB39). A form of profound prelingual sensorineural hearing loss. Sensorineural deafness results from damage to the neural receptors of the inner ear, the nerve pathways to the brain, or the area of the brain that receives sound information. Belongs to the peptidase S1 family. Plasminogen subfamily. 6 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Motility/polarity/chemotaxis; Hormone; Cell development/differentiation Chromosomal Location of Human Ortholog: 7q21.1 Cellular Component: extracellular space; membrane; extracellular region Molecular Function:identical protein binding; protein binding; growth factor activity; protein heterodimerization activity; serine-type endopeptidase activity; chemoattractant activity Biological Process: positive regulation of myelination; mitosis; platelet activation; organ regeneration; activation of MAPK activity; myoblast proliferation; hepatocyte growth factor receptor signaling pathway; negative regulation of caspase activity; proteolysis; liver development; positive regulation of phosphoinositide 3-kinase cascade; positive regulation of osteoblast differentiation; positive regulation of peptidyl-tyrosine phosphorylation; positive regulation of angiogenesis; platelet degranulation; positive chemotaxis; epithelial to mesenchymal transition; positive regulation of transcription from RNA polymerase II promoter; blood coagulation; hyaluronan metabolic process; positive regulation of cell migration Disease: Deafness, Autosomal Recessive 39
NCBI Summary:	Hepatocyte growth factor regulates cell growth, cell motility, and morphogenesis by activating a tyrosine kinase signaling cascade after binding to the proto-oncogenic c-Met receptor. Hepatocyte growth factor is secreted by mesenchymal cells and acts as a multi-functional cytokine on cells of mainly epithelial origin. Its ability to stimulate mitogenesis, cell motility, and matrix invasion gives it a central role in angiogenesis, tumorogenesis, and tissue regeneration. It is secreted as a single inactive polypeptide and is cleaved by serine proteases into a 69-kDa alpha-chain and 34-kDa beta-chain. A disulfide bond between the alpha and beta chains produces the active, heterodimeric molecule. The protein belongs to the plasminogen subfamily of S1 peptidases but has no detectable protease activity. Alternative splicing of this gene produces multiple transcript variants encoding different isoforms. [provided by RefSeq, Jul 2008]
UniProt Code:	P14210
NCBI GenInfo Identifier:	123116
NCBI Gene ID:	3082
NCBI Accession:	P14210.2
UniProt Secondary Accession:	P14210,Q02935, Q13494, Q14519, Q3KRB2, Q8TCE2, Q9BYL9 Q9BYM0, Q9UDU6, A1L3U6,
UniProt Related Accession:	P14210
Molecular Weight:	728
NCBI Full Name:	Hepatocyte growth factor
NCBI Synonym Full Names:	hepatocyte growth factor (hepapoietin A; scatter factor)
NCBI Official Symbol:	HGF
NCBI Official Synonym Symbols:	SF; HGFB; HPTA; F-TCF; DFNB39
NCBI Protein Information:	hepatocyte growth factor; hepatopoeitin-A; hepatopoietin-A; lung fibroblast-derived mitogen; fibroblast-derived tumor cytotoxic factor
UniProt Protein Name:	Hepatocyte growth factor
UniProt Synonym Protein Names:	Hepatopoietin-A; Scatter factor; SF
Protein Family:	Hepatocyte growth factor
UniProt Gene Name:	HGF
UniProt Entry Name:	HGF_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.

ELISA	Antibodies
Human HGF (Hepatocyte Growth Factor) CLIA Kit (HUES00081)	Anti-HGF Antibody (CAB1193)
Human HGF (Hepatocyte Growth Factor) ELISA Kit (HUES01370)	THBS2 Antibody (PACO07217)
	SCARB1 Antibody (PACO14504)
	HGF Antibody (PACO14505)
	CREB1 Antibody (PACO21100)