Protocol for Zymogram

Reagents:

1% Gelatin in H2O (Fisher Blood 275)

1% Casein (Sigma)

SDS-PAGE gel stock w/o urea

Wash buffer: 2.5% Triton X-100 in H2O (+0.02% NaN3)

Incubation Buffer: 50mM Tris-HCl (PH8.0), 5mM CaCl2, 0.02% NaN3

Gels:

Regular separating gel containing 10-12% substrate

Regular stacking gel

Protocol:

1- Collect media from cells

(if desired, inactivate non-metalloproteases with PMSF and/or NEM)

2- Centrifuge to remove cellular debris

(if necessary concentrate with centricon units or dialyze and lyophilize)

3- Add Laemmli loading buffer (OMIT UREA AND REDUCING AGENTS,

DO NOT HEAT)

4- Load samples directly onto gel

5- Run gel

6- Wash 2X 20min in wash buffer

7- Wash 10min in incubation buffer

8- Place gel in sealable container with fresh incubation buffer and incubate

at 37ºC for 24h to 48h

9- Fix and stain with fresh Coomassie Blue solution

10-Destain with MeOH:AcOH:H2O(5:1:5)

11-Replace with 10% AcOH and continue destaining

12-Photpgraph and dry gel for storage

(by Lee, Sunyoung)

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