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Extract collagen from rat tail (in lab)


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#1 Tran Nhat Anh

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Posted 28 October 2009 - 03:44 AM

I'm working on a project which try to produce an angiogenesis model in vitro. In my protocol, I have to mimic an extracellular matrix for my rat aortic rings with collagen type I. Does anybody know how to extract collagen from mammal skin? (like from rat tail or skin). Please tell me some methods to do that in an cell laboratory.

#2 badger

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Posted 03 November 2009 - 01:30 PM

I'm working on a project which try to produce an angiogenesis model in vitro. In my protocol, I have to mimic an extracellular matrix for my rat aortic rings with collagen type I. Does anybody know how to extract collagen from mammal skin? (like from rat tail or skin). Please tell me some methods to do that in an cell laboratory.



Hello!

I found the following protocol useful (although it's lengthy, it will give you plenty of collagen):

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 used 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




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