Custom Premix Selecting "Custom Premix" option means that all of the beads you have chosen will be premixed in manufacturing before the kit is sent to you.
Olson, M.W. et al. 2000. J. Biol. Chem.275, 2661. Kolkenbrock, H., et al. 1996. Biol. Chem. 377, 529.
数据表
Note that this data sheet is not lot-specific and is representative of the current specifications for this product. Please consult the vial label and the certificate of analysis for information on specific lots. Also note that shipping conditions may differ from storage conditions.
Revision
18-July-2007 RFH
Synonyms
Matrix Metalloproteinase 9, Gelatinase B, 92 kDa type IV Collagenase
Description
Dimeric, native MMP-9 from stimulated human neutrophils. Enzyme should be activated just prior to use.
Form
Liquid
Formulation
In 200 mM NaCl, 50 mM Tris-HCl, 5 mM CaCl₂, 1 µM ZnCl₂, 0.05% BRIJ® 35 Detergent, 0.05% NaN₃, pH 7.0.
Recommended reaction conditions
Organomercurial Activation ProtocolThis protocol is provided only as a general guide. Researchers should standardize this assay for their own specific needs and should consult published literature. The following protocol is from Stricklin, et al., which describes the use of p-aminophenylmercuric acetate (APMA) to activate pro-MMP. This protocol is also adaptable to other types of organomercurals, such as p-(hydroxymercuric) benzoate (PHMB), phenylmercuric chloride (PMC), or mersalyl.
1. Prepare a 10-50 mM stock solution of APMA (or other organomercurial compound) in 0.1 M NaOH just prior to use. Although not absolutely necessary, the stock solution may be adjusted to pH 11 with 5 N HCl (see Marcy, A.I., et al.).
2. To initiate the activation mix the proenzyme solution with the APMA solution at a 10:1 volume ratio (MMP:APMA). If a higher concentration of APMA is desired, increase the concentration of the stock solution. Do not exceed the 10:1 ratio, as this could result in significant changes in pH.
3. Incubate the mixture at 37°C for 2-3 h. It is recommended that an analytical run be conducted first to determine the optimal incubation time. For example, a small-scale experiment with a fixed concentration of pro-MMP and organomercurial would be incubated as described above. Remove aliquots of the sample at various time points during the incubation. Stop the reaction by the addition of SDS-PAGE sample buffer (e.g., 10 µl 2X sample buffer to 10 µl aliquot) and heat the samples to 95°C. The progress of activation can be monitored qualitatively by analyzing the aliquots on a 12% SDS-PAGE gel.
4. The activated MMP can be used without removing the APMA from the mixture. Please refer to Marcy, A.I., et al. for removal of organomercurials by gel filtration.
Source
Prepared from stimulated neutrophils that have been shown by certified tests to be negative for HBsAg and for antibodies to HIV and HCV.
EC number
3.4.24.35
Purity
≥90% by SDS-PAGE
Contaminants
No other MMP activity detectable
Specific activity
≥1000 mU/mg protein
Unit definition
One unit is defined as the amount of enzyme that will hydrolyze 1.0 µmol 2,4-DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg-OH per min at 37°C pH 7.0.
Storage
Avoid freeze/thaw
≤ -70°C
Do Not Freeze
Ok to freeze
Special Instructions
Following initial thaw, aliquot and freeze (-70°C).
Toxicity
Standard Handling
References
Olson, M.W. et al. 2000. J. Biol. Chem.275, 2661. Kolkenbrock, H., et al. 1996. Biol. Chem. 377, 529.
