more collagen extraction - (Jan/16/2008 )
the protocol am following to extract collagen (provided by chondrex) from fibroblast cell culture is as follows:
1: starting volume 0.05M acetic acid
2: 1/10 starting volume pepsin solution (1mg/ml in 0.05Macetic acid) incubation in 4c for 24-48 hours
3: 1/10 starting volume x10TBS (1.0M Tris-2.0M NaCl-50mM CaCl2, pH 7.8 - 8.0) adjust PH to 8
4: 1/10 starting volume of pancreatic elastase (1 mg/mL dissolved in 1X TBS, pH 7.8 - 8.0) incubate 24 hours in 4c
5: centrifuge at 10000 for 5 m at RT. and collect supernatant which will contain the soluble collagen
i have been trying this protocol with modifications for ages now, and never got any results on ELISA. and the company doesn't recommend any extra process to purify the collagen. but i read a lot of notes about salt precipitation process, i tried by adding 2M NaCl to a final concentration of 0.7M and centrifuge the next day and collect the pellet supposing to have my collagen there.
please collagen experts, what do you think? i think we should meet and put a united real proved protocol for collagen extraction and detection.
i appreciate your notes (again) !!!!
the modification i had was
1: to add blocking agent to the starting volume (goat serum 25%) to prevent collagen from sticking to the tube's wall
2: adjust NaCl concentration from 0.15 M to 0.3 M (claimed to prevent collagen from forming insoluble fibril (gel))
and i also wounder about the room temperature noted for the centrifugation step!!!
I got a new note that protein will not be precipitated by salt in a diluted solution !!! WHY??? i think i am missing some basics here