TIMP-2 Protein, Mouse (HEK293, His)

TIMP-2 is a tissue inhibitor of metalloproteinases that forms complexes with metalloproteinases, especially collagenases, effectively and irreversibly inactivating them by binding to their catalytic zinc cofactor.This interaction plays a crucial role in regulating the activity of metalloproteinases.TIMP-2 Protein, Mouse (HEK293, His) is the recombinant mouse-derived TIMP-2 protein, expressed by HEK293 , with C-6*His labeled tag.

Tissue inhibitor of metalloproteinases-2 (TIMP-2) is a protein that forms complexes with metalloproteinases, including collagenases, leading to the irreversible inactivation of these enzymes by binding to their catalytic zinc cofactor. Notably, TIMP-2 exhibits a specific interaction with matrix metalloproteinase 2 (MMP2) through its C-terminal region. This interaction, particularly with the C-terminal PEX domain of MMP2, results in the inhibition of MMP2 activity. TIMP-2's ability to regulate metalloproteinases underscores its significance in controlling extracellular matrix remodeling, with implications for various physiological and pathological processes. (

Measured by its ability to inhibit human MMP-2 cleavage of a fluorogenic peptide substrate Mca-PLGL-Dpa-AR-NH2. The IC₅₀ is 4.79 nM, as measured under the described conditions.

• 
  Measured in a cell proliferation assay using HUVEC human umbilical vein endothelial cells. The ED₅₀ this effect is 34.88 ng/mL, corresponding to a specific activity is 2.87×10⁴ units/mg.

Materials
Assay buffer: 50 mM Tris, 10 mM CaCl?, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
Test protein: TIMP-2 Protein, Mouse (HEK293, His) (HY-P71137)
Tool protein: MMP-2 Protein, Human (HEK293, His) (HY-P70268)
Activator for Human MMP-2: p-Aminophenylmercuric acetate (HY-148905, APAM), dissolved in 40 mM DMSO
Substrate: MOCAc-PLGL(Dpa)AR (HY-131498), dissolved in 2 mM DMSO

Procedure
1. Dilute Human MMP-2 to 100 μg/mL in assay buffer.
2. Add APMA to Human MMP-2 to a final concentration of 1 mM.
3. Incubate 100 μg/mL Human MMP-2 at 37°C for 1 hour to activate.
4. Dilute Mouse TIMP-2 protein with assay buffer to concentrations of 400 nmol/L, 200 nmol/L, 100 nmol/L, 50 nmol/L, 25 nmol/L, 12.5 nmol/L, 6.25 nmol/L, 3.13 nmol/L, and 1.56 nmol/L.
5. Dilute the Human MMP-2 activated with assay buffer to 2 μg/mL.
6. Experimental group: 20 μL of Mouse TIMP-2 protein at varying concentrations + 20 μL of 2 μg/mL Human MMP-2 protein;
Control group: 20 μL assay buffer + 20 μL, 2 μg/mL Human MMP-2 protein.
7. Incubate reaction mixtures at 37°C for 2 hours.
8. Dilute the reaction by adding 160 μL assay buffer.
9. Dilute the substrate to 20 μM in assay buffer.
10. Dispense 50 μL of the diluted mixture into a black microplate and add 50 μL substrate to initiate the reaction.
11. Read for 5 minutes in dynamic mode at excitation wavelength 320 nm and emission wavelength 405 nm.
12. Determine the 50% inhibitory concentration (IC₅₀) for Mouse TIMP-2 by plotting the RFU/min versus concentration relationship using 4-PL fitting.

Mouse

HEK293

C-6*His

P25785 (C27-P220)

21858  [NCBI]

Full Length of Mature Protein

CSCSPVHPQQAFCNADVVIRAKAVSEKEVDSGNDIYGNPIKRIQYEIKQIKMFKGPDKDIEFIYTAPSSAVCGVSLDVGGKKEYLIAGKAEGDGKMHITLCDFIVPWDTLSITQKKSLNHRYQMGCECKITRCPMIPCYISSPDECLWMDWVTEKSINGHQAKFFACIKRSDGSCAWYRGAAPPKQEFLDIEDP

Approximately 21 kDa, based on SDS-PAGE under reducing conditions.

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  ≥ 90%, as determined by reducing SDS-PAGE.

Solution.

Supplied as a 0.22 μm filtered solution of PBS, pH 7.4.

<1 EU/μg, determined by LAL method.

N/A.

Stored at -80°C for 1 year from date of receipt. It is stable at -20°C for 3 months after opening. It is recommended to freeze aliquots at -80°C for extended storage. Avoid repeated freeze-thaw cycles.

Shipping with dry ice.