Human Tryptase gamma / TPSG1 ELISA Kit

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SKU:
HUFI00928
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
Q9NRR2
Sensitivity:
18.75pg/ml
Range:
31.25-2000pg/ml
ELISA Type:
Sandwich ELISA, Double Antibody
Synonyms:
TPSG1, Tryptase gamma ?1, gamma I, gamma II, lung tryptase, mast cell protease II, mast cell tryptase, PRSS31skin tryptase, Serine protease 31, Transmembrane tryptase, trpA, tryptase gamma, tryptase gamma 1, tryptase gamma I, tryptase gamma II
Reactivity:
Human
Frequently bought together:

Description

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Human Tryptase gamma / TPSG1 ELISA Kit

Tryptases are a family of trypsin-like serine proteases in the S1 peptidase family. Only heparin-stabilized tetramers of tryptases are enzymatically active, and they are impervious to all known proteinase inhibitors. There are several tryptase genes on chromosome 16p13. There's a lot of debate about the number of genes in this cluster. Four useful genes - alpha I, beta I, beta II, and gamma I - have already been identified. Tryptase gamma 1/TPSG1 has a C-terminal hydrophobic domain, which may be used as a membrane anchor, unlike other members of the tryptase gene family. Tryptases have been linked to the development of asthma and other allergic and inflammatory diseases. Tryptophan metabolism disorders such as Hereditary Alpha Tryptasemia Syndrome and Gene Duplication Disease are linked to TPSG1.

Product Name:Human Tryptase gamma / TPSG1 ELISA Kit
Product Code:HUFI00928
Size:96 Assays
Alias:TPSG1, Tryptase gamma ?1, gamma I, gamma II, lung tryptase, mast cell protease II, mast cell tryptase, PRSS31skin tryptase, Serine protease 31, Transmembrane tryptase, trpA, tryptase gamma, tryptase gamma 1, tryptase gamma I, tryptase gamma II
Detection method:Sandwich ELISA, Double Antibody
Application:This immunoassay kit allows for the in vitro quantitative determination of Human TPSG1 concentrations in serum plasma and other biological fluids.
Sensitivity:18.75pg/ml
Range:31.25-2000pg/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery:Matrices listed below were spiked with certain level of Human TPSG1 and the recovery rates were calculated by comparing the measured value to the expected amount of Human TPSG1 in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)97-10099
EDTA plasma(n=5)88-10294
UFH plasma(n=5)86-9794
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human TPSG1 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)85-93%85-105%86-91%
EDTA plasma(n=5)84-95%84-100%85-99%
UFH plasma(n=5)81-95%82-100%80-95%
CV(%):Intra-Assay: CV<8%
Inter-Assay: CV<10%
ComponentQuantityStorage
ELISA Microplate (Dismountable)8×12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antibody(Concentrated)120ul4°C (Protect from light)
Antibody Dilution Buffer10ml4°C
HRP-Streptavidin Conjugate(SABC)120ul4°C (Protect from light)
SABC Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5-

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
UniprotQ9NRR2
UniProt Protein Function:TPSG1: Tryptases comprise a family of trypsin-like serine proteases, the peptidase family S1. Tryptases are enzymatically active only as heparin-stabilized tetramers, and they are resistant to all known endogenous proteinase inhibitors. Several tryptase genes are clustered on chromosome 16p13.3. There is uncertainty regarding the number of genes in this cluster. Currently four functional genes - alpha I, beta I, beta II and gamma I - have been identified. And beta I has an allelic variant named alpha II, beta II has an allelic variant beta III, also gamma I has an allelic variant gamma II. Beta tryptases appear to be the main isoenzymes expressed in mast cells; whereas in basophils, alpha-tryptases predominant. This gene differs from other members of the tryptase gene family in that it has C-terminal hydrophobic domain, which may serve as a membrane anchor. Tryptases have been implicated as mediators in the pathogenesis of asthma and other allergic and inflammatory disorders. [provided by RefSeq, Jul 2008]
UniProt Protein Details:Protein type:EC 3.4.21.-; Membrane protein, integral; Protease

Chromosomal Location of Human Ortholog: 16p13.3

Cellular Component: integral to plasma membrane

Molecular Function:serine-type endopeptidase activity; serine-type peptidase activity

Biological Process: proteolysis

NCBI Summary:Tryptases comprise a family of trypsin-like serine proteases, the peptidase family S1. Tryptases are enzymatically active only as heparin-stabilized tetramers, and they are resistant to all known endogenous proteinase inhibitors. Several tryptase genes are clustered on chromosome 16p13.3. There is uncertainty regarding the number of genes in this cluster. Currently four functional genes - alpha I, beta I, beta II and gamma I - have been identified. And beta I has an allelic variant named alpha II, beta II has an allelic variant beta III, also gamma I has an allelic variant gamma II. Beta tryptases appear to be the main isoenzymes expressed in mast cells; whereas in basophils, alpha-tryptases predominant. This gene differs from other members of the tryptase gene family in that it has C-terminal hydrophobic domain, which may serve as a membrane anchor. Tryptases have been implicated as mediators in the pathogenesis of asthma and other allergic and inflammatory disorders. [provided by RefSeq, Jul 2008]
UniProt Code:Q9NRR2
NCBI GenInfo Identifier:296453005
NCBI Gene ID:25823
NCBI Accession:Q9NRR2.3
UniProt Secondary Accession:Q9NRR2,Q96RZ8, Q9C015, Q9NRQ8, Q9UBB2,
UniProt Related Accession:Q9NRR2
Molecular Weight:33,815 Da
NCBI Full Name:Tryptase gamma
NCBI Synonym Full Names:tryptase gamma 1
NCBI Official Symbol:TPSG1  
NCBI Official Synonym Symbols:TMT; trpA; PRSS31  
NCBI Protein Information:tryptase gamma
UniProt Protein Name:Tryptase gamma
UniProt Synonym Protein Names:Serine protease 31; Transmembrane tryptase
Protein Family:Tryptase
UniProt Gene Name:TPSG1  
UniProt Entry Name:TRYG1_HUMAN

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

StepProtocol
1.Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.Aliquot 0.1ml standard solutions into the standard wells.
3.Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.Seal the plate with a cover and incubate at 37 °C for 90 min.
6.Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.Seal the plate with a cover and incubate at 37°C for 60 min.
9.Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14. Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

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 TypeProtocol
SerumIf 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.

PlasmaCollect 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 FluidCollect 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 supernatantCollect 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 lysatesSolubilize 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 homogenatesThe 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 lysatesRinse 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 MilkCollect 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.

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