I am trying to quantify/observe the expression(trend) of MMPs (matrix metalloproteinases) at different time points (i.e. day 0, 3, 5, 7 etc) from ASCs (Adipose derived stem cells) during osteogenic differentiation. To measure/observe the secreted MMPs (MMP-2, MMP-9, etc.), I would be performing western blot (WB) on the conditioned media (CM) collected at different time points. And to measure the membrane type MMPs (MT-MMP1), I would collect the cell lysate (in RIPA lysis buffer) at the corresponding time points and perform WB on them. So far so good...
However, I want to perform DNA assay as well (at the corresponding time points), so that I can normalize the MMP expression with respect to the no. of cells (DNA content) i.e. units of MMP/unit of DNA to have a meaningful comparison between different time points. Hence, I wanted to know if the same cell lysate (in RIPA lysis buffer) can be used to estimate the DNA content or do I have to make a separate group and perform DNA assay in TEXK lysing solution.
I would normalize for protein amount and use GAPDH, tubulin or actin as loading controls. I don't think RIPA lyses the nucleus and liberates the genomic DNA. However, that may depend on the specific recipe. If it would, you can't really pipette the solution due to high viscosity and you would need to treat the sample with ultrasonication or DNAse.
RIPA lysis buffer contains non-ionic and ionic detergents, which are able to extract protein from wide variety of cell types and membrane structures.
To normalize the expression of the MMPs I would go for a loading control as Dirk already suggested and then analyze the band size with Fiji or something else. If you are interested in the DNA amount at different time points, I would do a seperate group and isolate the DNA there, but for sure, you cannot compare it to the protein amount since it is a different experiment.
Dirk Hubmacher Yang Huanhuan Lena Brücker I thank you all for your feedbacks.
So just to summarize your suggestion: Units of MMP/units of GAPDH (tubulin or actin) is an alternative (better) metric to compare MMP secretion at different timepoints i.e. day 1, 3, 5, 7, 14 etc.?
Lena Brücker I am following this article: 10.1359/jbmr.2000.15.11.2154 where they compare the mmp expression of rat osteoblasts (Western Blots) at different time points. They mention that the mmp secreted across different groups (i.e. different time points) was from the same no. of cells (DNA content). How did they measure the DNA content at the same time, they must have done two separate assays right? I would really appreciate if you could help me with this.
Once again thanks everyone for your advices.
I skipped through the paper but I think they did two different sets of experiment, don't know how they can compare this then.. I would still go for loading the same protein amount and then put a loading control on the blot. I am sorry I cannot help you more. Maybe contact the authors?
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