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Index / Cell Sciences / Human MMP9 ELISA Kit / Product Detail : CKH176 Human MMP9 ELISA Kit
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#CKH176 Human MMP9 ELISA Kit

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Product name : Human MMP9 ELISA Kit

Catalog number : CKH176

Quantity: 1 x 96 tests

Availability: Yes

Supplier name : Cell Sciences

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About this Product :

Human MMP9 ELISA Kit
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More Details about

Human Matrix Metallopeptidase 9 ELISA Kit
Catalog No: CKH176 Size: 1 x 96 wells

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that degrade extracellular matrix proteins. MMPs have been linked with a wide array of biological activities and play important roles during organ development and pathological processes. Collectively MMPs are key enzymes for the metabolism of extracellular matrix proteins, including fibrillar and non-fibrillar collagens, fibronectin, laminin and basement membrane or interstitial stroma glycoproteins. Under physiological conditions MMPs are involved in extracellular degradation and breakdown of matrix proteins during normal tissue remodelling processes such as wound healing, pregnancy, and angiogenesis. Human MMP9 is a 92 kDa glycoprotein that plays a significant role in matrix remodeling, enzyme modulation, and cytokine/growth factor activation. MMP9 is also known as gelatinase B based on its ability to degrade gelatin.
The Cell Sciences Human MMP9 ELISA (Enzyme-Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of human MMP9 pro and active forms in serum, plasma (Collect plasma using heparin as an anticoagulant. EDTA and Citrate are not recommended), cell culture supernatants and urine. This assay employs an antibody specific for human MMP9 coated on a 96-well plate. Standards and samples are pipetted into the wells and MMP9 present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-human MMP9 antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of MMP9 bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.

Reagents and materials supplied with the kit:
Storage of Kit Reagents
Stable for up to 6 months from date of shipment at 2-4oC. Store reconstituted standard (recombinant protein) at -80oC. Opened Microplate Wells and reagents are stable for 1 month at 2-4oC. Return unused wells to the pouch containing desiccant pack and reseal along the entire edge. Kit is stable for one year if entire kit stored at -20oC.

Materials/reagents required but not provided:
• Microplate reader capable of measuring absorbance at 450 nm
• Precision pipettes to deliver 2 μl to 1 ml volumes
• Adjustable 1-25 ml pipettes for reagent preparation
• 100 ml and 1 liter graduated cylinders
• Absorbent paper
• Distilled or deionized water
• Log-log graph paper or computer and software for ELISA data analysis
• Tubes to prepare standard or sample dilutions
Preparation of Kit Reagents
1. Bring all reagents and samples to room temperature (18 - 25°C) before use.
2. Sample dilution: If your samples need to be diluted, Assay Diluent (Item E) is used for dilution of serum/plasma/culture supernatants/urine.
3. Assay Diluent (Item E) should be diluted 5-fold with deionized or distilled water before use.
4. Preparation of standard: Briefly spin the vial of Item C. Add 400 μl 1x Assay Diluent (Item E) into Item C vial to prepare a 50 ng/ml standard. Dissolve the powder thoroughly by a gentle mix. Add 80 μl MMP-9 standard from the vial of tem C, into a tube with 586.7 μl 1x Assay Diluent Buffer to prepare a 6000 pg/ml stock standard solution. Pipette 400μl 1x Assay Diluent into each tube. Use the stock standard solution to produce a Dilution series (shown below). Mix each tube thoroughly before the next transfer. Gently vortex to mix. 1x Assay Diluent serves as the zero standard (0 pg/ml).
5. If the Wash Concentrate (20x) (Item B) contains visible crystals, warm to room temperature and mix gently until dissolved. Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to yield 400 ml of 1x Wash Buffer.
6. Briefly spin the Detection Antibody vial (Item F) before use. Add 100 μl of 1x Assay Diluent into the vial to prepare a detection antibody concentrate. Pipette up and down to mix gently (the concentrate can be stored at 4°C for 5 days). The detection antibody concentrate should be diluted 100-fold with 1x Assay Diluent and used in step 4 of Part VI Assay Procedure.
7. Briefly spin the HRP-Streptavidin concentrate vial (Item G) and pipette up and down to mix gently before use. HRP-Streptavidin concentrate should be diluted 20,000-fold with 1x Assay Diluent. For example: Briefly spin the vial (Item G) and pipette up and down to mix gently . Add 2 μl of HRP-Streptavidin concentrate into a tube with 198.0 μl 1x Assay Diluent to prepare a 100-fold diluted HRP-Streptavidin solution (don’t store the diluted solution for next day use). Mix through and then pipette 70 μl of prepared 100-fold diluted solution into a tube with 14 ml 1x Assay Diluent to prepare a final 20,000 fold diluted HRP-Streptavidin solution.

ELISA Method
Be sure to read ‘Preparation of Kit Reagents’ before carrying out the assay.
1. Bring all reagents and samples to room temperature (18 - 25°C) before use. It is recommended that all standards and samples be run at least in duplicate.
2. Add 100 μl of each standard (see Preparation of Kit Reagents: MMP9 Standard) and sample into appropriate wells. Cover and incubate for 2.5 hours at room temp. or overnight at 2-4°C with gentle shaking.
3. Discard the solution and wash 4 times with 1x Wash Solution. Wash by filling each well with Wash Buffer (300 μl) using a multi-channel pipette or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining wash buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
4. Add 100 μl of 1x prepared biotinylated antibody (see Preparation of Kit Reagents: Detection Antibody) to each well. Incubate for 1 hour at room temperature with gentle shaking.
5. Discard the solution and wash as in step 3 above.
6. Add 100 μl of prepared Streptavidin solution (see Preparation of Kit Reagents: Streptavidin-HRP Concentrate) to each well. Incubate for 45 minutes at room temperature with gentle shaking.
7. Discard the solution and wash as in step 3 above.
8. Add 100 μl of TMB One-Step Substrate Reagent (Item H) to each well. Incubate for 30 minutes at room temperature in the dark with gentle shaking.
9. Add 50 μl of Stop Solution (Item I) to each well. Read at 450 nm immediately.

Assay Procedure Summary
1. Prepare all reagents, samples and standards as instructed.
2. Add 100 μl standard or sample to each well. Incubate 2.5 hours at room temperature, or over night at 4°C.
3. Add 100 μl prepared Biotin antibody to each well. Incubate 1 hour at room temperature.
4. Add 100 μl prepared Streptavidin solution. Incubate 45 minutes at room temperature.
5. Add 100 μl TMB One-step Substrate Reagent to each well. Incubate 30 minutes at room temperature.
6. Add 50 μl Stop Solution to each well. Read at 450 nm immediately.

Calculation of Results
Calculate the mean absorbance for each set of duplicate standards, controls and samples, and subtract the average zero standard optical density. Plot the standard curve on log-log graph paper or using plot software, with standard concentration on the x-axis and absorbance on the y-axis. Draw the best-fit straight line through the standard points.
These standard curves are for demonstration only. A standard curve must be run with each assay.
Performances and Characteristics
The minimum detectable dose of MMP9 is typically less than 10 pg/ml.
Recovery was determined by spiking various levels of human MMP9 into human serum, plasma and cell culture media. Mean recoveries are as follows:
Intra-assay: CV<10%
Inter-assay: CV<12%
Cross Reactivity: This ELISA kit shows no cross-reactivity with any of the cytokines tested (e.g., human Angiogenin, BDNF, BLC, ENA-78, FGF4, IL1α, IL1β, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12 p70, IL12 p40, IL13, IL15, IL309, IP10, G-CSF, GM-CSF, IFN-γ, Leptin (OB), MCP1, MCP3, MDC, MIP1α, MIP1 β, MIP1δ, MMP1, -2, -3, -10, PARC, RANTES, SCF, TARC, TGF-β, TIMP1, TIMP2, TNF-α, TNF-β, TPO, VEGF).

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