Biology Reference
In-Depth Information
Fig. 4. STEM analysis of macromolecular assemblies within the mass range
10-9000 kDa/nm. (A) Type VI collagen microfibrils, (B) fibrillin-containing
microfibrils. ( C and D ) Type I collagen fibrils of embryonic chick tendon and sea-
urchin ligament (sea-urchin ligament micrograph courtesy of Prof. J. Trotter, Albu-
querque, NM).
1.
Fibrillar collagen extraction buffer: 50 m M Tris-HCl (pH 7.4), 50 m M EDTA,
100 m M sucrose, 150 m M sodium chloride.
2.
Dounce homogenizer (Agar Scientific, Essex, U.K.).
3.
Inactive collagenase buffer: 50 m M Tris-HCl (pH 7.4), 0.4 M sodium chloride,
10 m M calcium chloride.
4.
Protease inhibitors: Prepare
100 stock of 2 m M phenylmethanesulphonyl fluo-
ride (PMSF) and 10 m M N-ethylmalemide (NEM) in methanol. Weigh out in a
fumehood or wear a face mask. Stock solution may be stored at 4
×
°
C for 4-5 d.
5.
Active collagenase buffer: to a 20-mL plastic universal tube add 10 mL collage-
nase buffer and 100 mL protease inhibitor stock. Weigh out 2 mg bacterial colla-
genase (type 1A) and add to mix to produce active collagenase buffer. The buffer
may be stored at 4
°
C for 4-5 d.
6.
Column buffer: 0.4 M sodium chloride, 50 m M Tris-HCl (pH 7.4).
7.
Sepharose CL-2B column: equilibrate 30 mL Sepharose CL-2B with column
buffer in a column of dimensions 1.5
×
25 cm at a flow rate of 0.2 mL/min overnight.
8.
Peristaltic pump, fraction collector, ultraviolet (UV) detector, chart recorder.
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