We digested off the cells first before lysing, which can then be centrifuged and lysing in a very small volume of Laemmli. If digesting off the cells first is not an option, I would use RIPA buffer instead Laemmli for lysis. Then concentrating the solution via freeze drying and solubilizing in Laemmli. In any case, some loss of material has to be accepted.

We lysate the cells directly on the TW, with a Lysing buffer: Tris 5 mM, NaCl 70 mM, Triton X-100 al 0.05%), 100 uL on the apical side, and shaking til the cells are floating. Then just collect them with the pipete, and complete the lysate with a 25G needle.

Hi David Schad

I'm lysing my transwell cultures for proteomics, but my method may still give you some ideas.

I'm using the 12mm transwells, after washing in PBS I peel the membrane from the support (could also be cut out). I then put half a membrane (or a whole 6.5mm membrane) in the bottom of a 1.5mL Eppendorf tube, and store them dry at -80.

When it comes time to lyse my cells (using 8M urea in my case), I just add the volume of lysis buffer to the Eppendorf and you can lyse on a rocker overnight, sonicate, vortex, or what ever works best for your type of cells. No problems with lysis buffer seeping through the membranes, because it's all contained in the Eppendorf.

Best of luck!

Sam