collagen extraction - (Oct/28/2009 )
i have asked this question many times ..
but i still havn`t arrived to the best way to extract collagen .. many protocols and many ways.
i will sum up my experience and please if any body has an opinion please share it..
collagen extraction from fibroblast growing in monolayer culture 10 cm flask.
the collagen can be found in two places
1: soluble collagen in the culture medium (no need for digestion u can process immediately for quantification, however the culture medium volume is to big that make collagen amount very diluted, so it need some kind of concentrating the collagen... and here where you can find several methods: 1) (I tried) salt precipitation by adding NaCl to 3M and ammonium sulfate to 30% incubate for 30 min at 4C , centrifuge at 10600 g for 10 min at 4 c and remove supernatant , the pellet contains the precipitated collagen dissolve in PBS. 2) ( i didnt try) lyophilize, and dissolve in PBS
2: the rest of the collagen is entrapped in the extracellular matrix, to collect scarp the cells with cell scraper (does it need cell lysing ? i think it does ) then digest the cell lysate with pepsin solution for 24 - 48 hours at 4C with rotating. (extra digestion with panceratic elastase is questionable, but seems to be important for some kind of antibodies that recognize special part of collagen that cant be exposed without elastase treatment) after pepsin digestion either process for quatification or precipitate collagen as mentioned above.
for quantification i use sircol red assay which quantify total collagen . for specific collagen type u can try ELISA (never succeed) or WB it works but there are many outcomes depend on the preparation and the antibody used , u can get signle band or two bands ( alpha one and alpha two ) or three bands (one alpha two and two alpha one) .
in conclusion i am still not confident when dealing with collagen is the most abundant protein in the body yet the most difficult to deal with .
abohollolo on Oct 28 2009, 06:20 AM said:
but i still havn`t arrived to the best way to extract collagen .. many protocols and many ways.
i will sum up my experience and please if any body has an opinion please share it..
collagen extraction from fibroblast growing in monolayer culture 10 cm flask.
the collagen can be found in two places
1: soluble collagen in the culture medium (no need for digestion u can process immediately for quantification, however the culture medium volume is to big that make collagen amount very diluted, so it need some kind of concentrating the collagen... and here where you can find several methods: 1) (I tried) salt precipitation by adding NaCl to 3M and ammonium sulfate to 30% incubate for 30 min at 4C , centrifuge at 10600 g for 10 min at 4 c and remove supernatant , the pellet contains the precipitated collagen dissolve in PBS. 2) ( i didnt try) lyophilize, and dissolve in PBS
2: the rest of the collagen is entrapped in the extracellular matrix, to collect scarp the cells with cell scraper (does it need cell lysing ? i think it does ) then digest the cell lysate with pepsin solution for 24 - 48 hours at 4C with rotating. (extra digestion with panceratic elastase is questionable, but seems to be important for some kind of antibodies that recognize special part of collagen that cant be exposed without elastase treatment) after pepsin digestion either process for quatification or precipitate collagen as mentioned above.
for quantification i use sircol red assay which quantify total collagen . for specific collagen type u can try ELISA (never succeed) or WB it works but there are many outcomes depend on the preparation and the antibody used , u can get signle band or two bands ( alpha one and alpha two ) or three bands (one alpha two and two alpha one) .
in conclusion i am still not confident when dealing with collagen is the most abundant protein in the body yet the most difficult to deal with .
Hello!
Try isolation of collagen type I from rat tails. Although it's a lengthy protocol , it gives you plenty of collagen that will last a long time. My last batch lasted a year. Alternatively, you can buy it.
Here is the protocol:
Important: ALWAYS keep the collagen solution cool, since it will otherwise solidify!
- Get 8-10 rat tails (they can have been stored at -20C) and disinfect them with 70% ethanol by submerging
- Remove and discard skin
- Remove tendons from the tail and place them in 70% ethanol for sterilization
- Weigh tendons and add pre-cooled 0.5 M acetic acid: 250 ml per 1g tendon, e.g. 2l for 8g
- stir at 4C for 48-72 hours
- Centrifuge at 4C and 10 000 g for 30 min and discard the pellet
- Add an equal volume of pre-cooled 10%(w/v) NaCl to the supernatant (e.g. 1.9l) for precipitation at 4C; I found it useful to precipitate over night and the next day the collagen floated on top so that it could be easily harvested
- collect collagen-rich insoluble material by centrifugation 30 min at 10 000 g and 4C
- discard supernatant and resuspend the pellet in 0.25M acetic acid at 4C (this may take a long time, e.g. overnight on a magnetic stirrer in a coldroom); avoid adding too much acetic acid as it will dilute the collagen (e.g. for 9g initial tendons, resuspend in 800 ml)
- dialyse against diluted acetic acid (1:1000; 1ml per l) at 4C for three days and change buffer twice a day; I fused a membrane for MW 12-14 000
- sterilize by centrifugation for 2 hours at 20 000 g (sterile filtration and heat are both no option for sterilization)
- storage in sterile bottle at 4C
- collagen solution can be diluted as required by adding sterile 1:1000 acetic acid
Good luck!