Protein Lysis of 3D Cell Culture in Matrigel Imbedded or On Top using PBS-EDTA buffer;
To 50mL of cold 1x PBS buffer add;
500 microliter of 0.5M EDTA to 5mM
250 microliter of 0.2M NaVO4 to 1mM
250 microliter of 0.25M NaF to 1.5mM
500 microliter of 100x Protease Inhibitor Cocktail to 1x
After taking pictures and rid of medium of the cell culture, for a 35 mm dish, rinse once quickly with about 2 mL of cold 1xPBS, and leave the dish on Ice
Add about 2-3 mL of PBS-EDTA on to the culture, use a polyethylene cell lifter or a p1000 pipet with tip cut off, pipet the cell/mtg mixture up and down to dislodge from the dish and transfer to a 15 mL conical tube
Repeat 2-3 times and use about total 10 mL of PBS-EDTA until all cell-gel mixture is transferred.
Shake the tubes in cold room or on ice for 45 min to1 hour to dissolve the mtg.
Centrifuge for 5 min at 1000rpm at 4C
Transfer the cell pellet to an 1.5mL eppendorf tube on ice* #, Centrifuge and rid of supernatant, add about 100-150 microliter of RIPA buffer. (adjust the volume of RIPA according to pellet size, concentrated protein solution is desired)
Sonicate on ice about 20 sec at setting @3-4
Centrifuge for 10 min at 14,000rpm at 4C
Transfer supernatant contains protein to another clean eppendorf tube, quick freeze on dried ice before stored in -80C freezer
*If matrigel is not all dissolved, try again with a few mL of PBS-EDTA , break the gel with the same pipet tip, invert the tube a few times before centrifuge, Do not require prolonged shaking this time.
# if there is a problem in dissolving the matrigel, check your EDTA concentration.