Hello everyone, I am designing an experiment to investigate cell secretion of MMPs. My main question is, after how long can one detect secreted protein? I have also observed that researcher either use medium or protein sample to carry out western blotting for MMPs. I would like to know which of these methods has the best results and why.
Thank you.
As an example, an attached fibroblast can make about 12 molecules of fibronectin per second, (based on calculations done in the mathematics in biology workshop at the Fields institute in Toronto back in 2008). So, once attached, cells make things happen quickly. The next question is, how much will actually matter in your assay, and if they are degraded, when do you hit steady state? Does tension promote cleavage? Etc. Not sure if this helps, but maybe provides a framework for consideration.
Well the time scale should be the same as for membrane turnaround, i.e., endocytotic and exocytotic vesicles fission and fusion events. Exact time will depend on the cell type. First secretion is of course visible after just part of that time scale.
Dear Lucy,
How long does it take for cells to secrete extracellular matrix proteins, is difficult to answer, it depends on your cell lines and your conditions. So I think you will have to make a time series (something like 2h, 4h, 8h, 16h, 24h and 48h, but it depends on your conditions) and make a western blot with the cell culture supernatants. Than you only have the secreted MMPs, if you use the cell extracts than you also measure the MMPs protein synthesis an the immobilized MMPs (at the cell membrane). If you use cell culture supernatant your MMPs will be diluted in the "big volume" of the media (so you get maybe a weak signal in the western blot).
Maybe the use of gel Zymography would also be fine for you, than you can examine the activity of your MMPs.
Good Luck
As an example, an attached fibroblast can make about 12 molecules of fibronectin per second, (based on calculations done in the mathematics in biology workshop at the Fields institute in Toronto back in 2008). So, once attached, cells make things happen quickly. The next question is, how much will actually matter in your assay, and if they are degraded, when do you hit steady state? Does tension promote cleavage? Etc. Not sure if this helps, but maybe provides a framework for consideration.
Hi,
you can use zymography, but this is not quantitative. the best to get quantitative info is ELISA or a bead based platform (such as luminex). western blot can be used, albumin may disturb the migration if you are using cells cultured in medium + serum...
as for the timing, I agree with the answers above, that a time course is the best approach to decide the time at which to collect your samples for subsequent analyses.
Thank you.Your suggestions have been very helpful. For the MMP, I wll try ELISA. For how long it takes to secret the protein, I will definitely design a time series. Once again i do appreciate the suggestions.
It is not clear which material do you want to use. We detected ECM proteins by WB after 24 h of in vitro culture .
10 fibronectin molecules per second might sound like a lot but it pales in comparison to the ~2000 antibody molecules/s secreted by mature b-cells http://www.ncbi.nlm.nih.gov/books/NBK26884/. So, not only the cells and conditions but the molecules should be considered. Your MMP secretion rates will likely need to be determined empirically. Also, because different MMPs interact differently with cells and ECM proteins, care must be taken in selecting samples (cells, medium, matrix) for your kinetic experiments.
You can also perform a proliferation assay with the exact cell amount you are going to use for MMPs assay. If you are using the supernatant to detect the secreted MMPs then you can relate to protein amount in the cell layer.
- Badges
- Science topic
- Similar topics
- Biological Science
- Cell Biology