Dominik Haudenschild August 10, 1993 at CBRC

1) Get sterna from 7-8 week old chicken (younger is better, less cross linking)

2) Remove perichondrium and bone

3) Freeze on dry ice and roughly homogenize in coffee grinder

4) Extract 1 to 2 hours (not longer) with 4M Guanidine, 50mM sodium acetate pH 5.8 on ice

5) Pellet solid material (collagen) and wash pellet 3-4 times with ice cold water

6) Homogenize in 20 volumes 0.5M acetic acid

7) Adjust pH to 2.5 with HCl

8) Add pepsin to 1mg/ml

9) Pepsin digest for 48 hours at 4°C

10) Pellet insoluble matter

11) Precipitate collagen from supernatant by adding solid NaCl to 0.7M

12) Centrifuge to collect precipitated collagen

13) Re dissolve collagen in 50mM Tris, 1M NaCl pH 7.4

14) Remove insoluble material by centrifugation

15) Precipitate collagen by adding dry salt to 4.0M

16 Centrifuge to collect precipitated collagen

17) Re dissolve pellet in 0.5M Acetic acid

18) Precipitate collagen by adding solid NaCl to 1.2M

19) Centrifuge to collect precipitated collagen

20) Re-dissolve in 0.5M Acetic Acid

21) Precipitate by complete dialysis against 20mM sodium Phosphate pH 7.4

22) Centrifuge to collect precipitated collagen

23) Resuspend in 0.1M acetic acid

24) Lyophilize