Human Serine protease HTRA1 (HTRA1) ELISA Kit

Product Name:	Human Serine protease HTRA1 (HTRA1) ELISA Kit
SKU:	HUEB2096
Size:	96T
Target:	Human Serine protease HTRA1 (HTRA1)
Synonyms:	High-temperature requirement A serine peptidase 1, L56, Serine protease 11, HTRA, PRSS11
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	78-5000pg/mL
Sensitivity:	20pg/mL

Intra CV:

5.3%

Inter CV:

6.3%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	108-120%	106-114%	91-100%	108-118%
EDTA Plasma(N=5)	96-106%	93-103%	80-92%	94-106%
Heparin Plasma(N=5)	100-110%	102-111%	107-117%	109-119%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	80	80-86
Plasma	82	80-88

Function:

Serine protease with a variety of targets, including extracellular matrix proteins such as fibronectin. HTRA1-generated fibronectin fragments further induce synovial cells to up-regulate MMP1 and MMP3 production. May also degrade proteoglycans, such as aggrecan, decorin and fibromodulin. Through cleavage of proteoglycans, may release soluble FGF-glycosaminoglycan complexes that promote the range and intensity of FGF signals in the extracellular space. Regulates the availability of insulin-like growth factors (IGFs) by cleaving IGF-binding proteins. Inhibits signaling mediated by TGF-beta family members. This activity requires the integrity of the catalytic site, although it is unclear whether TGF-beta proteins are themselves degraded. By acting on TGF-beta signaling, may regulate many physiological processes, including retinal angiogenesis and neuronal survival and maturation during development. Intracellularly, degrades TSC2, leading to the activation of TSC2 downstream targets.

Uniprot:	Q92743
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Serine protease HTRA1
Sub Unit:	Forms homotrimers. In the presence of substrate, may form higher-order multimers in a PDZ-independent manner. Interacts with TGF-beta family members, including BMP4, TGFB1, TGFB2, activin A and GDF5.
Research Area:	Signal Transduction
Subcellular Location:	Cell membrane Secreted Cytoplasm Cytosol Predominantly secreted (PubMed:15208355). Also found associated with the plasma membrane (PubMed:21297635).
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	HTRA1: Serine protease with a variety of targets, including extracellular matrix proteins such as fibronectin. HTRA1-generated fibronectin fragments further induce synovial cells to up-regulate MMP1 and MMP3 production. May also degrade proteoglycans, such as aggrecan, decorin and fibromodulin. Through cleavage of proteoglycans, may release soluble FGF-glycosaminoglycan complexes that promote the range and intensity of FGF signals in the extracellular space. Regulates the availability of insulin-like growth factors (IGFs) by cleaving IGF-binding proteins. Inhibits signaling mediated by TGF-beta family members. This activity requires the integrity of the catalytic site, although it is unclear whether TGF-beta proteins are themselves degraded. By acting on TGF-beta signaling, may regulate many physiological processes, including retinal angiogenesis and neuronal survival and maturation during development. Intracellularly, degrades TSC2, leading to the activation of TSC2 downstream targets. Variations in the promoter region of HTRA1 are the cause of susceptibility to age-related macular degeneration type 7 (ARMD7). ARMD is the leading cause of vision loss and blindness among older individuals in the developed word. It is classified as either dry (nonneovascular) or wet (neovascular). ARMD7 is a wet form, in which new blood vessels form and break beneath the retina. This leakage causes permanent damage to surrounding retinal tissue, distorting and destroying central vision. Wet ARMD is more prevalent among Asians than Caucasians. Defects in HTRA1 are the cause of cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL). CARASIL is characterized by nonhypertensive cerebral small-vessel arteriopathy with subcortical infarcts, alopecia, and spondylosis, with an onset in early adulthood. On neuropathological examination, atherosclerosis associated with intimal thickening and dense collagen fibers, loss of vascular smooth-muscle cells, and hyaline degeneration of the tunica media has been observed in cerebral small arteries. Belongs to the peptidase S1B family.
UniProt Protein Details:	Protein type:EC 3.4.21.-; Secreted, signal peptide; Secreted; Protease Chromosomal Location of Human Ortholog: 10q26.3 Cellular Component: extracellular matrix; extracellular space; cytosol Molecular Function:insulin-like growth factor binding; serine-type peptidase activity; serine-type endopeptidase activity Biological Process: negative regulation of defense response to virus; regulation of cell growth; negative regulation of transforming growth factor beta receptor signaling pathway; proteolysis; positive regulation of epithelial cell proliferation; negative regulation of BMP signaling pathway Disease: Macular Degeneration, Age-related, 7; Cerebral Autosomal Recessive Arteriopathy With Subcortical Infarcts And Leukoencephalopathy
NCBI Summary:	This gene encodes a member of the trypsin family of serine proteases. This protein is a secreted enzyme that is proposed to regulate the availability of insulin-like growth factors (IGFs) by cleaving IGF-binding proteins. It has also been suggested to be a regulator of cell growth. Variations in the promoter region of this gene are the cause of susceptibility to age-related macular degeneration type 7. [provided by RefSeq, Jul 2008]
UniProt Code:	Q92743
NCBI GenInfo Identifier:	18202620
NCBI Gene ID:	5654
NCBI Accession:	Q92743.1
UniProt Secondary Accession:	Q92743,Q9UNS5, D3DRE4,
UniProt Related Accession:	Q92743
Molecular Weight:
NCBI Full Name:	Serine protease HTRA1
NCBI Synonym Full Names:	HtrA serine peptidase 1
NCBI Official Symbol:	HTRA1
NCBI Official Synonym Symbols:	L56; HtrA; ARMD7; ORF480; PRSS11; CARASIL
NCBI Protein Information:	serine protease HTRA1; IGFBP5-protease; protease, serine, 11 (IGF binding); high-temperature requirement A serine peptidase 1
UniProt Protein Name:	Serine protease HTRA1
UniProt Synonym Protein Names:	High-temperature requirement A serine peptidase 1; L56; Serine protease 11
Protein Family:	Serine protease
UniProt Gene Name:	HTRA1
UniProt Entry Name:	HTRA1_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 HTRA1 / PRSS11 ELISA Kit	Anti-HTRA1 Antibody (CAB11693)
Human HTRA1 (HtrA Serine Peptidase 1) CLIA Kit (HUES00305)	HTR3A Antibody (PACO09839)
Human HTRA1 (HtrA Serine Peptidase 1) ELISA Kit (HUES01636)	NCLN Antibody, Biotin conjugated (PACO63834)
	HTRA1 Antibody (PACO63835)
	HTRA1 Antibody, HRP conjugated (PACO63836)