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Biotinylated MMP-9 His&Avi Tag Protein, Human

Biotinylated MMP-9 His&Avi Tag Protein, Human

Catalog Number: UA011192 Brand: UA BIOSCIENCE
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$552 USD
$552 USD
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Product Details

Product Specification


Species Human
Synonyms Type V Collagenase，Gelatinase B，EC 3.4.24.35，MANDP2，Matrix Metalloproteinase 9，CLG4B，MMP9，Matrix Metallopeptidase 9，Matrix Metalloproteinase 9 (Gelatinase B， 92kDa Gelatinase， 92kDa Type IV Collagenase)，GELB，Matrix Metalloproteinase-9，92 KDa Type IV Collagenase，Macrophage Gelatinase，92 kDa gelatinase，MMP-9，Matrix Metallopeptidase 9 (Gelatinase B， 92kDa Gelatinase， 92kDa Type IV Collagenase)，EC:3.4.24.35，EC 3.4.24
Accession P14780
Amino Acid Sequence

Ala20-Asp707 with His & Avi Tag at C-Terminus
Expression System HEK293
Molecular Weight

80-95kDa (Reducing)
Purity > 95 % as determined by SDS-PAGE & SEC-HPLC
Endotoxin <0.1EU/μg
Conjugation Biotin
Tag Avi Tag, His Tag
Physical Appearance Lyophilized Powder
Storage Buffer

PBS, PH7.4, 5% trehalose
Reconstitution

Reconstitute at 0.1-1 mg/ml according to the size in ultrapure water after rapid centrifugation.
Stability & Storage

· 12 months from date of receipt, lyophilized powder stored at -20 to -80℃. 
· 3 months, -20 to -80℃ under sterile conditions after reconstitution.
· 1 week, 2 to 8℃ under sterile conditions after reconstitution.  
· Please avoid repeated freeze-thaw cycles.
Reference

1.St-Pierre Y, et al. (2003) Emerging features in the regulation of MMP-9 gene expression for the development of novel molecular targets and therapeutic strategies. Curr Drug Targets Inflamm Allergy. 2(3): 206-15.
2.St-Pierre Y, et al. (2004) Regulation of MMP-9 gene expression for the development of novel molecular targets against cancer and inflammatory diseases. Expert Opin Ther Targets. 8(5): 473-89.
3.Chakrabarti S, et al. (2005) Matrix metalloproteinase-2 (MMP-2) and MMP-9 in pulmonary pathology. Exp Lung Res. 31(6): 599-621.

Background

Matrix metalloproteinases (MMPs) are neutral proteases involved in extracellular matrix (ECM) breakdown and remodeling during physiological processes like morphogenesis, angiogenesis, and tissue repair, as well as in pathological conditions such as inflammation, arthritis, and cancer. MMP9, or 92-kDa gelatinase B/type IV collagenase, is secreted by neutrophils, macrophages, and transformed cells. It is critical for tissue remodeling in inflammatory processes and is a key product of macrophages and neutrophils. MMP9 is also released by lymphocytes and stromal cells in response to inflammatory cytokines or integrin-mediated signals. Excessive MMP9 production is linked to tissue damage and degenerative disorders, making its regulation a focus for therapeutic development. Its overexpression in cancer and inflammatory conditions highlights the importance of understanding the mechanisms controlling its expression to identify potential therapeutic targets.

Protocol

Assay protocol

Principle: Measured by its ability to cleave a peptide substrate, Mca-PLGL-Dpa-AR-NH2.

Materials

  1. Assay Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij 35, pH 7.5 (TCNB)

  2. Biotinylated MMP-9 His&Avi Tag Protein, Human

  3. p-aminophenylmercuric acetate (APMA) (Aladdin, Catalog # P649286)

  4. Fluorogenic Peptide Substrate: MCA-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH2 (R&D, ES001)

  5. Calibration standard: MCA-Pro-Leu-OH (Shyuanye, T77046)

  6. 96 ELISA Removable Plate, Black, High binding (GENEVER, Catalog # GMO2-96H)

  7. Plate Reader (PerkinElmer, excitation 320 nm, and emission 405 nm)

Produce

  1. Activate MMP-9 at 100 μg/mL with 1 mM APMA in Assay Buffer. Incubate at 37 ℃ for 1 hour.

  2. Dilute activated MMP-9 (From the step 2) to 0.8, 0.4 and 0.2 μg/mL in Assay Buffer.

  3. Dilute Substrate to 20 μM in Assay Buffer.

  4. Load 50 μL of the dilute MMP-9 Protein (From the step 2) into a black well plate and start the reaction by adding 50 μL of 20 μM Substrate. Include a Substrate with Assay Buffer as Blank.

  5. Read at excitation and emission wavelengths of 320 nm and 405 nm, kinetic mode, 60s/cycle, 20 cycles.

  6. Calculate specific activity.

• Standard Curve

1. Dilute Calibration standard to 10 μM in Assay Buffer and prepare serial dilutions.

2. Add 100 μL of each serially diluted standard and blank (Assay buffer) into appropriate wells of a 96-well, the standard curve has a range of 1000, 500, 250, 125, 62.5, 31.25, 15.625 pmol per well.

3. Read at excitation and emission wavelengths of 320 nm and 405 nm, respectively in endpoint mode.

4. Linear Regression of MCA-Pro-Leu-OH (pmol)(y) – RFU-Blank(x).

Specific Activity (pmol/min/µg) =

Adjusted Vmax (RFU/min) x Conversion Factor (pmol/RFU)

amount of enzyme (µg)

 

Picture

SDS-PAGE

1μg (R: reducing condition, N: non-reducing condition).

SEC-HPLC

The purity of Biotinylated MMP-9 His&Avi Tag Protein, Human is greater than 95% as determined by SEC-HPLC.

ELISA

Immobilized Biotinylated MMP-9 His&Avi Tag Protein, Human (Cat. No. UA011192) at 2μg/mL on Streptavidin precoated (0.5μg/well) plate, can bind MMP9 Recombinant Rabbit mAb (SDT-368-16) (Cat. No. S0B2297) with EC50 of 16.52-36.26ng/ml.