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.
Search WWH ::
Custom Search