Detecting active MMP-9 levels

March 03, 2015

University of Southern California
Physiology and Biophysics
Graduate Student

Overall

Quality of Results

Ease-of-Optimization

What do these ratings mean?
Write a Review

Company:

AnaSpec

Product Name:

SensoLyte Plus 520 MMP-9 Assay Kit Fluorimetric and Enhanced Selectivity

Catalog Number:

72017

I am interested in detecting molecular biomarkers. I used this kit to detect active matrix metalloproteinase-9 (MMP-9) levels in human biofluids (e.g., cerebrospinal fluid). I am familiar with this company and this particular kit was well-reviewed and inexpensive. I am happy with products from this company and will continue to purchase from them.

Experimental Design and Results Summary

Application

Molecular markers

Starting Material

Human cerebrospinal fluid

Protocol Overview

For human cerebrospinal fluid (CSF) I used neat sample for this assay (depending on your starting material you may want to dilute your sample). Following desired sample dilutions and standard preparation, the samples/standards are added to the 96-well plate in duplicate. The plate incubates for 1 hour at room temperature and then is washed with wash buffer. To measure activity, MMP-9 substrate 5-FAM/QXL peptide 520 is added to each well. For end-point reading, the plate incubates at room temperature in the dark for 1 hour to 16 hours (since the concentration of active MMP-9 is low in human CSF, I incubated the plate for 16 hours). Then fluorescence intensity is measured on a plate reader at Ex/Em = 490nm/520nm. Concentrations are calculated from the fluorescence signal reading.

Tips

I recommend shaking the plate on a microtiter plate shaker during the incubation steps.

Results Summary

The concentration of the unknown samples are calculated from the known standard concentrations. The samples and standards are run in duplicate alongside each other during the assay to ensure accuracy of the measurements.

Additional Notes

The image provided was published in supplemental figure S4 in: "Montagne A, Barnes SR, Sweeney MD, Halliday MR, Sagare AP, Zhao Z et al. Blood-brain barrier breakdown in the aging human hippocampus. Neuron 2015; 85: 296–302."

Related Categories

Image Gallery

Summary

The Good

Easy to follow protocol.

The Bad

It would be better if the range of detection were broader for measuring active MMP-9 levels. However this is one of the most sensitive kits currently available.

The Bottom Line

This kit is one of few commercially available to measure active MMP-9 concentrations in human samples. I'm happy with this company and will continue to purchase this product.

Join the discussion