Use Collagenase Type VII (Sigma C-0773)

Make 3 units/µl stock in Milli-Q, stable -70°C for >6 months

Enzyme runs as many bands, 4 major bands, all <116kd

Digestion Conditions:
50mM Tris pH 7.5
25mM CaCl2 (if using protease inhibitors, must be non-chelating)
37°C for 5 hours or longer
Protein at ______ µg/ml
Collagenase at ______µg/ml

Use Chondroitinase ABC protease free (Seikagaku 100332)

Enzyme runs at ~80kd. Make 10U/ml stock, store -70°C

Digestion Conditions:
100mM Tris pH 8.0
30mM Sodium Acetate
37°C for 1 hour
Use 0.02 units per µg protein

Use Hyaluronidase SD (Seikagaku 100741)

Enzyme runs at ~125kd (gel filtration). Make 50mM phosphate pH 6.2 + 0.01% BSA to dissolve protein

Digestion Conditions:
40mM phosphate buffer pH 6.2
0.01% BSA
37°C for 10 minutes, heat inactivate for 1 minute at 100°C