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Rat Total MMP-9 Quantikine ELISA Kit

  • Assay Type
    Solid Phase Sandwich ELISA
  • Format
    96-well strip plate
  • Assay Length
    4.5 hours
  • Sensitivity
    0.028 ng/mL
  • Assay Range
    0.156 - 10 ng/mL
  • Specificity
    Natural and recombinant rat MMP-9 (Pro-, active, and TIMP-complexed)
  • Cross-reactivity
    < 0.5% cross-reactivity observed with available related molecules.< 50% cross-species reactivity observed with species tested.
  • Interference
    No significant interference observed with available related molecules.
Product Summary
The Quantikine Rat Total MMP-9 Immunoassay is a 4.5 hour solid phase ELISA designed to measure total rat MMP-9 (Pro-, active, and TIMP-complexed MMP-9) in cell culture supernates, serum, and plasma. It contains NS0-expressed recombinant rat MMP-9 and antibodies raised against the recombinant factor. This immunoassay has been shown to accurately quantitate the recombinant protein. Results obtained using natural rat MMP-9 showed dose-response curves that were parallel to the standard curves obtained using the Quantikine kit standards. These results indicate that this kit can be used to determine relative mass values for natural rat MMP-9.

Precision
Intra-Assay Precision (Precision within an assay) Three samples of known concentration were tested in separate assays to assess inter-assay precision.
Inter-Assay Precision (Precision between assays Three samples of known concentration were tested on one plate to assess intra-assay precision.
Cell Culture Supernates, Serum, Platelet-poor Heparin Plasma
Intra-Assay Precision Inter-Assay Precision
Sample123123
n202020202020
Mean0.391.225.270.511.183.55
Standard Deviation0.0270.0680.2380.0350.0460.153
CV%6.95.64.56.93.94.3

Recovery

The recovery of rat MMP-9 spiked into various matrices was evaluated.

Sample Type Average % Recovery Range %
Cell Culture Samples (n=4) 94 87-107
Platelet-poor Heparin Plasma (n=4) 92 80-103
Serum (n=4) 97 90-115
Linearity
To assess the linearity of the assay, samples containing high concentrations of rat MMP-9 were serially diluted with Calibrator Diluent to produce samples with values within the dynamic range of the assay.
Rat Total MMP-9 Quantikine ELISA Kit
Background: MMP-9

The matrix metalloproteinases (MMPs) consist of 24 known human zinc proteases with essential roles in breaking down components of the extracellular matrix (ECM). Additional MMP substrates include cytokines, chemokines, growth factors and binding proteins, cell/cell adhesion molecules, and other proteinases. With a few exceptions, MMPs share common structural motifs including a pro-peptide domain, a catalytic domain, a hinge region, and a hemopexin-like domain. Synthesized as pro-enzymes, most MMPs are secreted before conversion to their active form. MMP activities are modulated on several levels including transcription, pro-enzyme activation, or by their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs). A subset of MMPs are associated with membranes and designated as membrane-type metalloproteinases (MT-MMP).

    • Long Name
      Matrix Metalloproteinase 9
    • Entrez Gene IDs
      4318 (Human); 17395 (Mouse); 81687 (Rat);
    • Alternate Names
      CLG4B; Gelatinase B; GELB; 92 kDa gelatinase; 92 kDa type IV collagenase; CLG4B; EC 3.4.24; EC 3.4.24.35; Gelatinase B; GELB; macrophage gelatinase; MANDP2; matrix metallopeptidase 9; matrix metalloproteinase 9; matrix metalloproteinase-9; MMP-9; type V collagenase;
    Related Research Areas
    Assay Procedure
    Refer to the product for complete assay procedure.

    Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate.
    1.   Prepare all reagents, standard dilutions, and samples as directed in the product insert.
    2.   Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.

    3. 50 µL Assay Diluent
    4.   Add 50 µL of Assay Diluent to each well.

    5. 50 µL Standard, Control, or Sample
    6.   Add 50 µL of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 2 hours.
    7.   Aspirate each well and wash, repeating the process 4 times for a total of 5 washes.

    8. 100 µL Conjugate
    9.   Add 100 µL of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 2 hours.
    10.   Aspirate and wash 5 times.

    11. 100 µL Substrate Solution
    12.   Add 100 µL Substrate Solution to each well. Incubate at room temperature for 30 minutes. PROTECT FROM LIGHT.

    13. 100 µL Stop Solution
    14.   Add 100 µL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.

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