Human MMP 9 ELISA Kit

SKU:
HUFI00211
€299

Description

ELISA Kit Technical ManualMSDS

Human MMP-9 (Matrix Metalloproteinase 9) ELISA Kit - Information

The Assay Genie Human MMP-9 (Matrix Metalloproteinase 9) ELISA Kit can assay for Human MMP-9 in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.

How our Human MMP-9 (Matrix Metalloproteinase 9) 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 Human MMP-9 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 MMP-9 (Matrix Metalloproteinase 9) ELISA Kit Data

Product Code

HUFI00211

Uniprot P14780
Alias

MMP-9, Matrix Metalloproteinase 9, CLG4B, Gelatinase B, GELB, CLG4B, macrophage gelatinase, MANDP2, MMP-9, type V collagenase

Detection method

Sandwich ELISA, Double Antibody

Application

This immunoassay kit allows for the in vitro quantitative determination of Human MMP-9 concentrations in serum plasma and other biological fluids.

Size

96T

Range

0.313-20ng/ml

Sensitivity

0.188ng/ml

Storage

4'C for 6 months

Recovery

Matrices listed below were spiked with certain level of Human MMP-9 and the recovery rates were calculated by comparing the measured value to the expected amount of Human MMP-9 in samples.

MatrixRecovery range(%)Average(%)
serum(n=5) 85-97 91
EDTA plasma(n=5) 88-103 96
UFH plasma(n=5) 91-104 100
Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human MMP-9 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample1:21:41:8
serum(n=5) 85-104% 85-101% 85-105%
EDTA plasma(n=5) 87-101% 84-101% 82-88%
UFH plasma(n=5) 83-98% 92-100% 85-99%
CV(%)

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Note

For Research Use Only

Human MMP-9 (Matrix Metalloproteinase 9) ELISA Kit Protocol

The below protocol is a sample protocol for Human MMP-9 (Matrix Metalloproteinase 9) ELISA Kit using a biotinylated detection antibody and streptavidin-HRP. Sandwich ELISA Kits 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 MMP-9 Antibody 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.

Sandwich Protocol

Sandwich ELISA Protocol

Assay 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 MMP-9 (Matrix Metalloproteinase 9) 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 MMP-9 (Matrix Metalloproteinase 9) 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 MMP-9 Protein Information

UniProt Protein Function: MMP9: May play an essential role in local proteolysis of the extracellular matrix and in leukocyte migration. Could play a role in bone osteoclastic resorption. Cleaves KiSS1 at a Gly-|-Leu bond. Cleaves type IV and type V collagen into large C-terminal three quarter fragments and shorter N-terminal one quarter fragments. Degrades fibronectin but not laminin or Pz-peptide. Exists as monomer or homodimer; disulfide-linked. Exists also as heterodimer with a 25 kDa protein. Macrophages and transformed cell lines produce only the monomeric form. Interacts with ECM1. Activated by 4-aminophenylmercuric acetate and phorbol ester. Up-regulated by ARHGEF4, SPATA13 and APC via the JNK signaling pathway in colorectal tumor cells. Produced by normal alveolar macrophages and granulocytes. Inhibited by histatin-3 1/24 (histatin-5). Inhibited by ECM1. Belongs to the peptidase M10A family.
UniProt Protein Details:

Protein type:Protease; Secreted, signal peptide; Secreted; Motility/polarity/chemotaxis; EC 3.4.24.35

Chromosomal Location of Human Ortholog: 20q13.12

Cellular Component: extracellular space; proteinaceous extracellular matrix; extracellular region

Molecular Function:collagen binding; identical protein binding; protein binding; zinc ion binding; metalloendopeptidase activity; endopeptidase activity

Biological Process: positive regulation of keratinocyte migration; extracellular matrix disassembly; collagen catabolic process; axon guidance; extracellular matrix organization and biogenesis; ossification; macrophage differentiation; positive regulation of apoptosis; ephrin receptor signaling pathway; proteolysis; leukocyte migration; skeletal development; embryo implantation

Disease: Metaphyseal Anadysplasia 2

NCBI Summary: Proteins of the matrix metalloproteinase (MMP) family are involved in the breakdown of extracellular matrix in normal physiological processes, such as embryonic development, reproduction, and tissue remodeling, as well as in disease processes, such as arthritis and metastasis. Most MMP's are secreted as inactive proproteins which are activated when cleaved by extracellular proteinases. The enzyme encoded by this gene degrades type IV and V collagens. Studies in rhesus monkeys suggest that the enzyme is involved in IL-8-induced mobilization of hematopoietic progenitor cells from bone marrow, and murine studies suggest a role in tumor-associated tissue remodeling. [provided by RefSeq, Jul 2008]
UniProt Code: P14780
NCBI GenInfo Identifier: 269849668
NCBI Gene ID: 4318
NCBI Accession: P14780.3
UniProt Secondary Accession: P14780,Q3LR70, Q8N725, Q9H4Z1, Q9UCJ9, Q9UCL1, Q9UDK2 B2R7V9,
UniProt Related Accession: P14780
Molecular Weight: 78,458 Da
NCBI Full Name: Matrix metalloproteinase-9
NCBI Synonym Full Names: matrix metallopeptidase 9 (gelatinase B, 92kDa gelatinase, 92kDa type IV collagenase)
NCBI Official Symbol: MMP9  
NCBI Official Synonym Symbols: GELB; CLG4B; MMP-9; MANDP2  
NCBI Protein Information: matrix metalloproteinase-9; 92 kDa gelatinase; type V collagenase; macrophage gelatinase; 92 kDa type IV collagenase; matrix metalloproteinase 9 (gelatinase B, 92kDa gelatinase, 92kDa type IV collagenase)
UniProt Protein Name: Matrix metalloproteinase-9
UniProt Synonym Protein Names: 92 kDa gelatinase; 92 kDa type IV collagenase; Gelatinase B; GELB
Protein Family: Matrix metalloproteinase
UniProt Gene Name: MMP9  
UniProt Entry Name: MMP9_HUMAN

Human MMP 9 ELISA Kit Citations

Larkin et al. Role of matrix metalloproteinases 2 and 9, toll-like receptor 4 and platelet-leukocyte aggregate formation in sepsis-associated thrombocytopenia PLoS One. (2018) PubMed: 29734352

Citations

Larkin et al. Role of matrix metalloproteinases 2 and 9, toll-like receptor 4 and platelet-leukocyte aggregate formation in sepsis-associated thrombocytopenia PLoS One. (2018) PubMed: 29734352
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
Sandwich
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