Recombinant Human TIMP-4 (carrier free)

Product Details

Source

Human TIMP-4, amino acids Cys30-Pro224 (Accession # Q99727) with a C-terminal TG-8H-GGQ tag was expressed in CHO cells.

Molecular Mass

The 208 amino acid recombinant protein has a predicted molecular mass of approximately 23.9 kD. The non-reduced and DTT-reduced proteins migrate at 24 and 25 kD by SDS-PAGE, respectively.

Purity

> 95%, as determined by Coomasie stained SDS-PAGE

Formulation

0.22 µm filtered protein solution is in 20 mM TRIS, 300 mM NaCl, pH 7.5.

Endotoxin Level

Less than 1.0 EU per µg of protein as determined by the LAL method

Concentration

10 and 25 µg sizes are bottled at 200 µg/mL. 100 µg size and larger sizes are lot-specific and bottled at the concentration indicated on the vial. To obtain lot-specific concentration and expiration, please enter the lot number in our Certificate of Analysis online tool.

Storage & Handling

Unopened vial can be stored between 2°C and 8°C for up to 2 weeks, at -20°C for up to six months, or at -70°C or colder until the expiration date. For maximum results, quick spin vial prior to opening. The protein can be aliquoted and stored at -20°C or colder. Stock solutions can also be prepared at 50 - 100 µg/mL in appropriate sterile buffer, carrier protein such as 0.2 - 1% BSA or HSA can be added when preparing the stock solution. Aliquots can be stored between 2°C and 8°C for up to one week and stored at -20°C or colder for up to 3 months. Avoid repeated freeze/thaw cycles.

Activity

The activity of hTIMP-4 was measured by its ability to inhibit mature hMMP-2. IC₅₀ Value of hTIMP-4 is less than 1.4 nM (34 ng/mL).

Application

Bioassay

Application Notes

Human TIMP-4 Activity assay

 

Human TIMP-4 activity is measured by its ability to inhibit cleavage of a fluorogenic peptide substrate Mca-PLGL(Dpa)AR by activated hMMP-2. The potency of hTIMP-4 is described as IC₅₀ value.

 

Materials

1. Assay Buffer: TCNB (50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij-35, pH 7.5)
2. Recombinant Human MMP-2 (Predicted MW:  73.2 kDa)
3. Recombinant human TIMP-4 (Predicted MW: 23.9 kDa)
4. MMP-2 fluorogenic substrate: Mca-PLGL(Dpa)AR
5. P-Aminophenylmercuric acetate (APMA)

 

Activity assay procedures

1. Dilute hMMP-2 in the Assay buffer at 100 µg/mL.
2. Activate hMMP-2 by adding APMA to a final concentration of 1 mM.
3. Incubate the activating mixture for 1 hours at 37 °C.
4. Dilute the activated hMMP-2 to 4 ug/mL (0.4 ng/µL) in Assay Buffer.
5. Make serial dilution of hTIMP-4 in Assay Buffer from 16000 ng/mL to 7.81 ng/mL, and include 0 ng/mL as control.
6. Add into a tube 25 µL of each serially-diluted hTIMP-4 including the control, and then add 25 µL of 4 µg/mL activated hMMP-2.
7. Incubate the mixture at 37 °C for 2 hours.
8. Add 200 µL of Assay Buffer into each tube prepared in step 6 to make an assay mixture.
9. Dilute the substrate in Assay Buffer at 20 µM.
10. Load into a well plate 50 μL of each assay mixture and start the reaction by adding 50 μL of 20 μM Substrate. Include a substrate blank containing 50 μL Assay Buffer and 50 μL of 20 μM Substrate.
11. Read at excitation and emission wavelengths of 320 nm and 405 nm, respectively, in kinetic mode for 5 minutes.
12. Analyze the data with non-linear regression to estimate IC₅₀ value of hTIMP-4 towards hMMP-2.

 

Final assay conditions per well

• hMMP-2: 20 ng (200 ng/mL)
• hTIMP-2: 800, 400, 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.57, 0.781, 0 ng/mL
• Substrate: 10 µM

BioLegend carrier-free recombinant proteins provided in liquid format are shipped on blue-ice. Our comparison testing data indicates that when handled and stored as recommended, the liquid format has equal or better stability and shelf-life compared to commercially available lyophilized proteins after reconstitution. Our liquid proteins are verified in-house to maintain activity after shipping on blue ice and are backed by our 100% satisfaction guarantee. If you have any concerns, contact us at tech@biolegend.com.

Antigen Details

Structure

Monomer

Distribution

Brain, adipose tissue, Kidney, and heart; NTERA-2, U-87 MG,  and A549 cell line

Function

Regulation of Matrix Metalloproteinase activity

Interaction

MMPs

Ligand/Receptor

MMP-2

Bioactivity

Regulate MMP activities

Biology Area

Angiogenesis, Apoptosis/Tumor Suppressors/Cell Death, Cell Adhesion, Cell Biology

Molecular Family

Enzymes and Regulators

Antigen References

1. Chaturvedi P, Tyagi S. C.  2016 J Cell Mol Med 20:2089
2. Liss M. et al. 2009 Am J Pathology 175:940
3. Melendez-Zajgla J. et al. 2008 Mol Cancer 7:85
4. Page-McCaw A. et al. 2007 Nat Rev Mol Cell Biol 8:221
5. Groft et al. 2001 Br J Cancer 85:55.
6. Greene J. et al. 1996 J Biol Chem 271:30375-80

Gene ID

7079 View all products for this Gene ID

UniProt

View information about TIMP-4 on UniProt.org

Documentation

• Technical Data Sheet (pdf)
• Certificate of Analysis
• Material Safety Data Sheet

Reviews

Related Pages & Pathways

Related FAQs

Why choose BioLegend recombinant proteins?

     • Each lot of product is quality-tested for bioactivity as indicated on the data sheet.
     • Greater than 95% Purity or higher, tested on every lot of product.
     • 100% Satisfaction Guarantee for quality performance, stability, and consistency.
     • Ready-to-use liquid format saves time and reduces challenges associated with reconstitution.
     • Bulk and customization available. Contact us.
     • Learn more about our Recombinant Proteins.

How does the activity of your recombinant proteins compare to competitors?

We quality control each and every lot of recombinant protein. Not only do we check its bioactivity, but we also compare it against other commercially available recombinant proteins. We make sure each recombinant protein’s activity is at least as good as or better than the competition’s. In order to provide you with the best possible product, we ensure that our testing process is rigorous and thorough. If you’re curious and eager to make the switch to BioLegend recombinants, contact your sales representative today!

What is the specific activity or ED50 of my recombinant protein?

The specific activity range of the protein is indicated on the product datasheets. Because the exact activity values on a per unit basis can largely fluctuate depending on a number of factors, including the nature of the assay, cell density, age of cells/passage number, culture media used, and end user technique, the specific activity is best defined as a range and we guarantee the specific activity of all our lots will be within the range indicated on the datasheet. Please note this only applies to recombinants labeled for use in bioassays. ELISA standard recombinant proteins are not recommended for bioassay usage as they are not tested for these applications.

Have your recombinants been tested for stability?

Our testing shows that the recombinant proteins are able to withstand room temperature for a week without losing activity. In addition the recombinant proteins were also found to withstand four cycles of freeze and thaw without losing activity.

Does specific activity of a recombinant protein vary between lots?

Specific activity will vary for each lot and for the type of experiment that is done to validate it, but all passed lots will have activity within the established ED50 range for the product and we guarantee that our products will have lot-to-lot consistency. Please conduct an experiment-specific validation to find the optimal ED50 for your system.

How do you convert activity as an ED50 in ng/ml to a specific activity in Units/mg?

Use formula Specific activity (Units/mg) = 10^6/ ED50 (ng/mL)

Certificate of Analysis