Human Tryptase alpha / TPS1 ELISA Kit
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주문- SKU:
- HUFI00943
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Uniprot:
- Q15661
- Sensitivity:
- 0.938ng/ml
- Range:
- 1.563-100ng/ml
- ELISA Type:
- Sandwich ELISA, Double Antibody
- Synonyms:
- Tryptase alpha, TPS1, TPSAB1, Tryptase 1, TPS2, TPSB1, tryptase alpha, beta 1
- Reactivity:
- Human
Description
Human Tryptase alpha / TPS1 ELISA Kit
Tryptases are members of the trypsin-like serine protease family, which is known as the S1 peptidase family. Only heparin-stabilized tetramers of tryptases are enzymatically active, and they are resistant to all known proteinases inhibitors. Tryptases have been linked to the development of asthma and other allergic and inflammatory diseases. Matrix Metalloproteinases and Extracellular Matrix Degradation are two pathways that are associated with Tryptase alpha/TPS1.
Human Tryptase alpha / TPS1 ELISA Kit - Information
The Assay Genie Tryptase alpha / TPS1 ELISA Kit can assay for Tryptase alpha / TPS1 in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.
How our Tryptase alpha / TPS1 ELISA Kits Work?The Assay Genie (enzyme-linked immunosorbent assays) assay kits are designed for the quantitative measurement of analytes in a wide variety of samples. As today's scientists demand high quality consistent data for high impact journals, Assay Genie have developed our range of sensitive, fast and reliable ELISA kit assays to meet and exceed those demands. Our assay kits use a quantitative sandwich ELISA technique and each kit comes with highly specific antibodies pre-coated onto a 96-well microtiter plate.
At Assay Genie we understand the need for speed! Therefore, we have developed an ultra-fast protocol meaning you achieve your results rapidly. So, once you have prepared and plated your samples, blanks and standards, you simply incubate with a highly specific biotin-conjugated primary antibody and Avidin conjugated to Horseradish Peroxidase (HRP) and incubate for the appropriate length of time. After washing the plate according to the protocol and addition of the TMB (3,3',5,5'-Tetramethylbenzidine) solution, the appearance of a blue colour should be detected due to an enzymatic reaction catalysed by HRP. Next step is the addition of the Stop Solution which terminates the HRP reaction and the blue colour turns yellow with the signal intensity measured on a plate reader at 450nm. The amount of bound Tryptase alpha / TPS1 is proportional to the signal generated by the reaction meaning the kit assay gives you a quantitative measurement of the analyte in your samples.
Human Tryptase alpha / TPS1 ELISA Kit - Data
| Description | Tryptase is the major neutral protease present in mast cells and is secreted upon the coupled activation-degranulation response of this cell type. May play a role in innate immunity. Isoform 2 cleaves large substrates, such as fibronectin, more efficiently than isoform 1, but seems less efficient toward small substrates (PubMed:18854315). | |
| Post-Translational Modification | ||
| Uniprot ID | Q15661 |
| Alias | Tryptase alpha/TPS1/TPSAB1/Tryptase 1/TPS2/TPSB1/tryptase alphabeta 1 | ||||||||||||||||||||
| Detection method | Sandwich ELISA Double Antibody | ||||||||||||||||||||
| Application | This immunoassay kit allows for the in vitro quantitative determination of TPSAB1 concentrations in serum plasma and other biological fluids. | ||||||||||||||||||||
| Size | 96T | ||||||||||||||||||||
| Range | 1.56-100ng/ml | ||||||||||||||||||||
| Sensitivity | < 0.938ng/ml | ||||||||||||||||||||
| Storage | 4'C for 6 months | ||||||||||||||||||||
| Recovery | Matrices listed below were spiked with certain level of TPSAB1 and the recovery rates were calculated by comparing the measured value to the expected amount of TPSAB1 in samples.
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| Linearity | The linearity of the kit was assayed by testing samples spiked with appropriate concentration of TPSAB1 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
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| CV(%) | Intra-Assay: CV<8% | ||||||||||||||||||||
| Note | For Research Use Only |
Human Tryptase alpha / TPS1 ELISA Kit Protocol
The below protocol is a sample protocol for Human Tryptase alpha / TPS1 ELISA Kit using a biotinylated detection antibody and streptavidin-HRP. Sandwich ELISAs allow for the detection and quantification of an analyte in a sample by using known analyte concentrations as standards and plotting absorbance of known concentrations vs known standard concentrations. This allows the researcher to calculate the amount of Human Tryptase alpha / TPS1 present in their sample.
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.
Kit Protocol:
| 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. |
Human Tryptase alpha / TPS1 ELISA Kit components | 96 Assays | Storage |
| ELISA Microplate(Dismountable) | 8×12 strips | 4°C for 6 months |
| Lyophilized Standard | 2 | 4°C/-20°C |
| Sample/Standard Dilution Buffer | 20ml | 4°C |
| Biotin-labeled Antibody(Concentrated) | 120ul | 4°C (Protect from light) |
| Antibody Dilution Buffer | 10ml | 4°C |
| HRP-Streptavidin Conjugate(SABC) | 120ul | 4°C (Protect from light) |
| SABC Dilution Buffer | 10ml | 4°C |
| TMB Substrate | 10ml | 4°C (Protect from light) |
| Stop Solution | 10ml | 4°C |
| Wash Buffer(25X) | 30ml | 4°C |
| Plate Sealer | 5 | - |
Other materials and equipment required:
The Assay Genie Human Tryptase alpha / TPS1 ELISA Kit will require other equipment and materials to carry out the assay. Please see list below for further details.- 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
Sample Preparation
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. |
Human Tryptase alpha / TPS1 ELISA Kit Protein Information
| UniProt Protein Function: | TPSAB1: Tryptase is the major neutral protease present in mast cells and is secreted upon the coupled activation-degranulation response of this cell type. Belongs to the peptidase S1 family. Tryptase subfamily. 2 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Secreted, signal peptide; EC 3.4.21.59; Protease; Secreted Chromosomal Location of Human Ortholog: 16p13.3 Cellular Component: extracellular matrix; extracellular space; extracellular region Molecular Function:protein binding; serine-type peptidase activity; serine-type endopeptidase activity Biological Process: extracellular matrix disassembly; extracellular matrix organization and biogenesis; defense response; 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. These genes are characterized by several distinct features. They have a highly conserved 3' UTR and contain tandem repeat sequences at the 5' flank and 3' UTR which are thought to play a role in regulation of the mRNA stability. These genes have an intron immediately upstream of the initiator Met codon, which separates the site of transcription initiation from protein coding sequence. This feature is characteristic of tryptases but is unusual in other genes. The alleles of this gene exhibit an unusual amount of sequence variation, such that the alleles were once thought to represent two separate genes, alpha and beta 1. Beta tryptases appear to be the main isoenzymes expressed in mast cells; whereas in basophils, alpha tryptases predominate. Tryptases have been implicated as mediators in the pathogenesis of asthma and other allergic and inflammatory disorders. [provided by RefSeq, Jul 2008] |
| UniProt Code: | Q15661 |
| NCBI GenInfo Identifier: | 18202508 |
| NCBI Gene ID: | 7177 |
| NCBI Accession: | Q15661.1 |
| UniProt Secondary Accession: | Q15661,P15157, Q15663, Q6B052, Q9H2Y4, Q9H2Y5, Q9UQI1 D2E6R9, D2E6S1, |
| UniProt Related Accession: | Q15661 |
| Molecular Weight: | Observed MW: 31kDaCalculated MW: 29kDa/30kDa |
| NCBI Full Name: | Tryptase alpha/beta-1 |
| NCBI Synonym Full Names: | tryptase alpha/beta 1 |
| NCBI Official Symbol: | TPSAB1 |
| NCBI Official Synonym Symbols: | TPS1; TPS2; TPSB1 |
| NCBI Protein Information: | tryptase alpha/beta-1; tryptase I; tryptase-1; tryptase-I; tryptase-III; tryptase beta 1; tryptase beta I; tryptase beta-1; tryptase alpha-1; tryptase alpha II; mast cell beta I tryptase; mast cell alpha II tryptase |
| UniProt Protein Name: | Tryptase alpha/beta-1 |
| UniProt Synonym Protein Names: | Tryptase I; Tryptase alpha-1 |
| Protein Family: | Tryptase |
| UniProt Gene Name: | TPSAB1 |
| UniProt Entry Name: | TRYB1_HUMAN |
