Bone Collagen type I - extraction (Sep/05/2006 )

Hi!

Does anyone has a good protocol to extract collagen type I from human bone?
I want collagen with a good quality so I can proceed with my experiments.

Please help me!

Thanks!

pals

---

First you have to grind/homogenize the bone, for instance in a freezer mill or chop it into very small pieces. Then put the pieces in 4M guanidine-HCl, 0.05M Tris-HCl pH=7.5 for 2 days at 4 degrees, preferebally shaking.
Centrifuge it and add 0.2M NaCl, 0.5M HOAc to the pellet and add pepsin to 1mg/ml, o/n at 4 degrees, while shaken.
Centrifuge and adjust the supernatant to pH=8 (inactivation of the pepsin).
Dialyse the supernatant over night at 4°C against 0.7M NaCl, 0.5M HOAc to selectively precipitate type I, II and III collagen.
Suspend the precipitate in 0.5M NaCl/ 50mM Tris-HCl pH=7.4 (discard insoluble material by centrifugation) and dialyse over night against 0.5M NaCl/ 50mM Tris-HCl pH=7.4. Add NaCl to 1.7M to precipitate collagen type III. Add NaCl to the supernatant to 2.6M to precipitate collagen type I.
You first have to precipitate the type III, if you go immediately for 2.6M NaCl, you'll get a mixture of both types.
You can dissolve it in 0.5M HOAc.
If you want it unsalted, you have to dialyze it again for 4 days at 4 degrees against 0.1M HOAc.

---

Do you have one for skin collagen as well? Thanks!

---

Thanks aspergillie for your reply. But I still have some questions....
1. I read that after having chopped the bone into small pieces, we need to deffat with acetone and wash with ethanol... Have you done this? Is it necessary?
2. In the centrifugation steps, what's the speed and time.
3. Can you quantify the collagen you isollated?
4. In the end, do you have a "nice" quantity of collagen? Can it be hidrolized with collagenase afterwards?

Thanks in advance. Sorry of any mistakes, english is not my natural language

pals

P.S. - Next week I'll try your protocol, then I'll tell you how it worked.

---

I've never tried it from bone, but this is a more general procedure. May be you have to add for bone things extra like defatting.
About the centrifugation, I don't know it exactly. I always do this at 4 degrees, about 30 min. Before the precipitation, I spun as fast as possible, depending on the tubes and centrifuge present.
During the precipitation, you don't shake anymore, so it is not necessary to spin it very fast.
I never tried a real quantification, but I always put some on SDS-PAGE to see if it has went correct. If there's a lot of type II present, it tends to coprecipitate (reprecipitation of your samples can help). But off course in bone is alot of type I.
And the quantity depends on the starting meterial, the more yoy put in, the more you'll get out. I always had "normal amounts", like 100 mg. But I don't know if it will go the same for bone.
I never gave it a collagenase threatment, but I cannot think of any reason why it should not work.
There is a very old publication about this in Methods in enzymology, Volume 82
pp. 3-913 (1982) Structural and Contractile Proteins Part A: Extracellular Matrix [2] Preparation and characterization of the different types of collagen
Pages 33-64
Edward J. Miller and R. Kent Rhodes
And an updated version in volume 144.

---

Hi aspergillie,

I'm sorry to bother you again, but I'm performing the protocol you gave me, and I have a question: in the precipitation steps is it visible any precipitate? Because I don't see anything.... Maybe it depends on the starting material (I started with 1g of bone). Or maybe it's normal not to see anything.... Can you help me?

Thanks

Pals

---

It does indeed depend on the starting material. As I've said, I've never worked with bone. For meniscus, I also cannot see a pellet. But for articulair cartilage, I get some gelly (gelatine?) like pellet, for annulus it's a little white one.
Resuspent in a little bit of HOAc, you can always dilute it further.
Also try the precipitate with 4.0M NaCl, normally you get some type II with it, but since there is way more type I in bone, maybe it's possible to also get it in this fraction. (The method works really good for type II, but I can also get type I)

---

Hi!

Maybe that's the case, the precipitate might be a little bit transparent, so it may be difficult to see... I'll run a gel and see if I really managed to extract collagen I...
Wish me luck!
Thanks for all your help!
Pals

---

I hope it!!! Good luck

---