Right now I am using 1% NP40 containing lysis buffer to collect the lysate from rabbit corneal fibroblast cells.
Hi Abirami, perlecan and nidogen are usually not cell associated, so they will not be solubilized with non-ionic detergents like NP-40.
These proteins are underneath the cells (if they make an ECM) and they will be difficult to solubilize because they are arranged and probably crosslinked.
You can try to add SDS-PAGE sample buffer to the wells that you have solubilized with NP-40. The cells are gone, but the ECM might still be there.
The best way to analyze ECM proteins from cultured fibroblast is using the supernatant of the cells. For that purpose, you set the cells for 24 hours serum free and the next day you take the supernatants. With that you make a TCA precipitation and separate the proteins with a 4-12 SDS-PAGE. That´s how we look for production of nidogen, laminin and so on in cultured fibroblasts of the skin.
I would agree with the collection of media and that will give you secreted. You can also do a urea extraction for the cell associated but as someone said before your protocol will not yield them.\ as they wont be soluble.
Check the antibodies as you may need to remove GAG chains so run the gel and prepare half in the presence of specific lyases and the other half without and run -+.... and then probe with ab.
You might also want to test a Guanidine Hydrochloride buffer, which works nicely for ECM protein extraction from tissues. To solubilize them, a urea+Thiourea+CHAPs buffer might work. Good luck with the experiments.
Thank you all for the valuable suggestions. I can understand that most of the ECM proteins are secreted, i just was thinking i can trap some synthesized protein from the cell and i think it's a little amount in the given time and hence i am not able to capture them. I will surely try the sample buffer treatment as well as collecting culture medium and precpitating with TCA. Hopefully i will get a better way soon to detect them. Really all your comments are so helpful.
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