Hi,
I am trying to study the expression profile of matrix metalloproteinase (MMP-2) from human mesenchymal stem cells (hMSCs) in an osteogenic pathway. I have conditioned media (CM) collected from monolayer osteogenic differentiation of hMSCs, pass them through centrifuge filters and run them through 10% Novex Zymogram gels and incubate the gels in Coomassie stain and scan the gels. I analyse the bands using Image J software.
My technique: I convert the 600 dpi scan of gels into 8-bit images and from the "Gel" module I select the bands and use "Plot bands" to get the pixel intensity map of the bands and estimate the area under the peaks. However, the pro-MMP2 (~72kDa) and MMP2 (~62kDa) are not well resolved and the peaks overlap. I can't denconvolute the peaks and hence estimate the individual contribution of pro-MMP2 and MMP2. Can someone help me quantifying both the bands? I would really appreciate your help.
I have attached pics to give some idea about my protocol. i am following the article: "Detection of Functional Matrix Metalloproteinases by Zympgraphy" ; doi: 10.3791/2445
Hi Srujan Singh . You could try taking images of the gel, cropping out the pro and active bands and import those images into Image J for quantitation.
Hi Srujan,
I agree with the above post - cropping the areas of the respective bands using ImageJ (select rectangles; Strg M; select Integrated Density values for analysis) would make it easier for you to quantify your data.
Anyhow you should try to resolve the pro-MMP2/MMP2 forms a little more (change gel percentage, use a gradient gel?) - or maybe just run the gel a little bit longer? Using the crop-version for analysis will not help you with any potential signal cross-talk you have from the two different gelatinases.
Best regards,
Christian
PS: I couldn't upload my graph - here are the values I got from your data (MMP2_Original; lanes 1 - 6):
proMMP2: 23647 24752 25953 33119 37560 40625
MMP2: 24018 23933 23947 25567 27503 32013
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