How can I isolate ECM protein from a 6 well dish? - (May/24/2010 )
Hi all,
I have a 6 well dish and my protein of interest is produced by the cells but is transorted to the ECM outside the cells. I wanted to differentiate between the amount produced by the cells and located still in the cells and the protein located outside of the cells. Is there way to isolate in a first step just the cells and then in a second step the ECM that should still be in the well in order to see differenced?
thank you all
Hi,
Not sure if this will work. You will have to find papers (or google) to get more info.
I would suggest treatment with trypsin (or other enzymes or combination thereof) to "separate" cells from ECM. As comparison, you would need to prepare trypsin treatment at low and high concentration (or short or long time), somewhat similar to MNase digestion. Wash cells several time with PBS to remove ECM. Then, add lysis buffer to lyse the cell pellet. This would be the intracellular protein of interest (iPOI).
Next, prepare total POI (tPOI, e.g. intra- and extra-cellular protein) on another plate. This could be done by adding lysis buffer directly (after PBS rinsing) to well to lyse the cells and solubilize the ECM.
Cheers,
lsek on May 25 2010, 05:38 AM said:
Not sure if this will work. You will have to find papers (or google) to get more info.
I would suggest treatment with trypsin (or other enzymes or combination thereof) to "separate" cells from ECM. As comparison, you would need to prepare trypsin treatment at low and high concentration (or short or long time), somewhat similar to MNase digestion. Wash cells several time with PBS to remove ECM. Then, add lysis buffer to lyse the cell pellet. This would be the intracellular protein of interest (iPOI).
Next, prepare total POI (tPOI, e.g. intra- and extra-cellular protein) on another plate. This could be done by adding lysis buffer directly (after PBS rinsing) to well to lyse the cells and solubilize the ECM.
Cheers,
Hey thanks for the info.
I have not been able to find any relevant papers online that is why I posted it on the forum.
I see what you are saying and I have also thought of ot myseld, but trypsin would also solubilise part of the ECM so I will be collectin that also. Unless when I spin the cells down the ECM will stay as supernatant... I am not sure
Found this paper. Not sure it is of use to you.
http://www.springerlink.com/content/g7t8462027120g73/
lsek on May 30 2010, 10:08 AM said:
http://www.springerlink.com/content/g7t8462027120g73/
Hey great, thank you very much, I will definatelly read it and make the best out of it
Cheers