1) Dissect sternal cartilage from 17 to 18 day chicken
Cut into caudal and cephalic part, discarding the middle part.
Put into saline on ice. All subsequent steps done at 4°C

2) Homogenize with polytron in:
(Solution #1) 1M NaCl
50mM Tris-HCl pH 7.4
1mM PMSF
100mM 6-Aminohexanoic acid
10mM N-Ethyl maleimide
10mM Disodium EDTA

3) Extract 20 hours at 4°C on a nutator

4) Centrifuge at 27kg (=26k rpm in TLA100.3 rotor, =23k with TCA 45)
at 4°C for 30 minutes

5) Take supernatant, centrifuge 115kg (=53k rpm TLA-100.3, =45k rpm in TCA 45) at 4°C for 2 hours

6) Discard supernatant and re suspend pellet in:
(Solution #2) 8M de ionized urea
150mM NaCl
50mM Tris-HCl pH 7.4
Use vigorous vortexing to get pellet into solution

7) Centrifuge 115kg (=53k rpm TLA-100.3, =45k rpm in TCA 45) at 4°C for 2 hours

8) Repeat step 6 and 7

9) Suspend final pellet (fibrils) is suspended in:
(Solution #3) 150mM NaCl
50mM Tris-HCl pH 8.0