protein extraction from transwell inserts - (Mar/06/2012 )
I am trying to establish a co-culture system of lung epithelial cells and fibroblasts using transwells with pore diameter 1μΜ and 0.4μΜ. I wonder if there is a different protocol for lysis of the cells grown on inserts.
In the case of plain cell culture dishes i used to wash the cells twice with ice cold PBS and then extract the total proteins by scrapping using the appropriate lysis buffer. Can I use the scrapper on the membrane of the insert or there is a danger to damage it? Is it possible to loose any buffer or protein extract through the transwell pores?
I would appreciate any advice very much!
You should be able to trypsinise the cells on the insert and lift them off that way. There is the possibility that you will get some leakage through the membrane, but this would require positive pressure on the filter - like you would apply if filtering something with a syringe and filter.
Or perhaps you can wash twice with PBS, scrape first, transfer cells to a centrifuge tube or some other container then add lysis buffer .