if you want to determine the level of collagen, simply lysed the cells with standard cell lysis buffer along with protease inhibitors. Go for western with collagen specific (type I or II or others) antibodies. We used colagen type I and III from Rockland.
Hello,
as Sudhiranjan mentioned you can directly lyse cells e.g. in 4% SDS, 0.1M DTT, sonicate briefly to reduce viscosity due to DNA, heat for 10 min at 70 deg C and run on a gel and perform western blot.
Have you also considered quantifying the collagen that is in the medium? Performing western blot against collagen in a cell pellet would only quantify collagen that is in the secretory pathway and well attached to the cell surface. At the same time there must be collagen proteins that are secreted in the medium but do not incorporate in the cellular ECM.
Measuring the collagen in the medium and/or in the medium is an easy task as suggested. But, does anyone have a solution on how to quantify collagen specifically secreted and embedded in the ECM?
Hello Luca,
You can try to perform cell shaving with trypsin and subsequent protein identification via mass spectrometry (MS). There would be a few tricky points in this procedure: ECM can be incompletely digested or if the digestion is to long cells would start to die due to anoikis or cell lysis. The data can be used for relative quantification of most identified proteins.
Alternatively you can try to isolate plasma membrane sheets with the associated ECM and then perform WB or MS analysis. Useful controls would be 1) quantification of collagen in whole cells, 2) quantification of collagen in plasma membrane / ECM enriched sample 3) quantification of collagen in plasma mambrane/ ECM depleted sample.
It would be quite useful to have some visual conformation of the existence of cell attached ECM in your cell culture and relative amount of collagen in it before quantification. Electron microscopy can give you both types of information when combined with gold coated collagen antibodies.
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