Hi everyone,
I've been decellularizing 2D cell cultures for immunos and LC-MS/MS using 20 mM NH4OH. Basically, I incubate my cells with this solution for 5 min and keep the ECM that is attached to the dishes. For immunos is quite simple, as I basically fix it in PFA and it's ready to go. For LC-MS/MS I used an SDS-PAGE sample buffer and scratched the samples with it to recover as much as possible. For these assays, no problem at all.
Now I want to make hydrogels out of it and for that I need to solubilize them first. Here is where my problems start. I've tried incubating the plates with 2M urea buffer in a 0,15 M NaCl 0,05 M Tris HCl pH 7,4 buffer with 1% protease inhibitors at 4 ºC in the dark for 1h (scratching the plates before and after incubation) but haven't been able to get any protein when quantifying it (BCA, I've checked and this kit is compatible). Is there any tip/suggestion you could share with me? Is there something I'm doing wrong? Any help is deeply appreciated :)
Note: I'm now going to try and leave the samples at 4 ºC for 2 days to see if that helps.
Thanks a lot in advance!
ECM is mainly sugars. I'm not sure how to make hydrogels out of them, but solubilising them will require breaking the long chain sugars (hyaluronic acid, chondroitin, etc.)
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