Review text: Ab 125181 is the purified preparation of the recombinant human matrix metalloproteinase 2 proform (rhMMP-2) consisting of amino acids 30- 660 fused with a His tag at the C-terminus. Biological activity of this preparation has thus far been measured using the colorimetric assay based on its ability to cleave the colorimetric peptide substrate, Mca-PLGL-DpaAR-NH2. We tested the biological activity of rhMMP-2 using gelatin, the natural substrate of this enzyme (1).
Detection of gelatinolytic activity of the recombinant human (rhMMP2) was performed essentially using minor modifications of methods described in the literature (2, 3). Briefly, about 10 ng of rhMMP-2 in 25 µl of 25 mM Tris-glycine buffer pH 6.8 was mixed with equal volume of 2 x SDS-sample buffer without ß-mercaptoethanol and incubated at 370C for 10 minutes. Samples were loaded on a 10 % acrylamide -Tris-glycine gel containing 0.1% gelatin and electrophoresed using Tris-glycine pH 8.3 containing 0.1% SDS. Electrophoresis was performed at 90 volts for 120 minutes. Immediately after electrophoresis, the gel was washed by gentle shaking and incubation in 40-50 ml of 2.5% Triton X-100 for 15 minutes. Washing step was repeated 3 more times. Subsequently, the gel was immersed in 40-50 ml of 50 mM Tris- Glycine pH 7.5, 100 mM NaCl, 4 mM CaCl2 and 0.02% Triton X-100 and incubated at 370C for 16 hours. For detection of the gelatinolytic activity, the gel was stained with 0.05% Commassie Blue for 1 hour, followed by imaging on the ChemiDoc MP Imaging System.
As seen from Figure 1, the rhMMP-2 sample produced a prominent geletinolytic band corresponding to a molecular mass of ~ 68 kDa. An additional, faint gelatinolytic band, corresponding to a molecular mass ~ 45 kDa also appears to be present in this preparation. Nonetheless, these data confirm the gelatinolytic activity of rhMMP-2. Furthermore, the C-terminal His-Tag does not interfere with the gelatinolytic activity of the recombinant protein.
Figure 1. Gelatinolytic activity of rhMMP-2 (Ab 151866)
Snoek-van Beurden, P. and Von den Hoff, J. (2005) BioTechniques 38, 73-83
Mannello, F. and Sebastiani M. (2003) Clinical Chem. 49, 1546-50
Ratnikov, B. Deryugina, E., and Strongin, A. (2002) Laboratory Investigation, 82, 1583-1590
Sample: Human Recombinant protein
Primary antibody (in addition to 'MMP2 protein (Active)')
Primary antibody: None used
Secondary antibody: None used
Mr. Pramod Mahajan
Submitted Jan 08 2013