【资源】Protocol of Gelatin Zymography
丁香园论坛
1450
Gelatin Zymography Protocol
1. Prepare gels (7.5%, 10% or 12%) according to the standard procedure. When preparing the running gel add gelatin stock solution (10 mg/ml in H2O) to get the gelatin concentration of 0.1% (1 mg/ml).
2. Mix one part sample with one part Tris-Glycine SDS Sample Buffer (2x) and let stand 10 minutes at room temperature. DO NOT HEAT.
3. Apply samples (typically 10-25 μg) and run the gel with 1x Tris-Glycine SDS Running Buffer according to the standard running conditions (~125V, constant voltage). Run time (60-120 min) will depend on the gel percentage and running buffer concentration and pH. The run is complete when the bromophenol blue tracking dye reaches the bottom of the gel.
4. After running, dilute the Zymogram Renaturing Buffer (10x) 1:9 with deionized water and incubate the gel in the buffer (100 ml for one or two mini-gels) with gentle agitation for 30 minutes at room temperature.
5. Decant the Zymogram Renaturing Buffer and replace with 1x Zymogram Developing Buffer (100 ml for one or two mini-gels). Equilibrate the gel for 30 minutes at room temperature with gentle agitation then replace with fresh 1x Zymogram Developing Buffer and incubate at 37℃ for at least four hours. Incubate overnight for maximum sensitivity. Incubation time can be reduced to as little as one hour for concentrated samples. The optimal result can be determined empirically by varying the sample load or incubation time.
6. Stain with Coomassie Blue R-250 for 30 minutes. For maximum contrast, use a stain concentration of 0.5% (w/v) instead of the usual concentration of 0.1%. Gels should be destained with an appropriate Coomassie R-250 destaining solution (Methanol : Acetic acid : Water (50 : 10 : 40). Areas of protease activity will appear as clear bands against a dark blue background where the protease has digested the substrate.
Sample Buffer (2X)
0.5 M Tris-HCl, pH 6.8 2.5 ml
Glycerol 2.0 ml
10% (w/v) SDS 4.0 ml
0.1% Bromophenol Blue 0.5 ml
Distilled Water to 10.0 ml
Running Buffer (10x) (1X)
Tris Base 29 g 2.9 g
Glycine 144 g 14.4 g
SDS 10 g 1.0 g
Distilled Water to 1 L to 1 L
pH=8.3
Renaturing Buffer (10x)
Triton X-100, 25% (v/v) in water
Developing Buffer
(10x) (1X)
Tris base 12.1 g 50mM
Tris-HCl 63.0 g 50mM
NaCl 117 g 0.2M
CaCl2 7.4 g 5 mM
Brij 35 0.2% 0.02%
Distilled Water to 1 L
1. Prepare gels (7.5%, 10% or 12%) according to the standard procedure. When preparing the running gel add gelatin stock solution (10 mg/ml in H2O) to get the gelatin concentration of 0.1% (1 mg/ml).
2. Mix one part sample with one part Tris-Glycine SDS Sample Buffer (2x) and let stand 10 minutes at room temperature. DO NOT HEAT.
3. Apply samples (typically 10-25 μg) and run the gel with 1x Tris-Glycine SDS Running Buffer according to the standard running conditions (~125V, constant voltage). Run time (60-120 min) will depend on the gel percentage and running buffer concentration and pH. The run is complete when the bromophenol blue tracking dye reaches the bottom of the gel.
4. After running, dilute the Zymogram Renaturing Buffer (10x) 1:9 with deionized water and incubate the gel in the buffer (100 ml for one or two mini-gels) with gentle agitation for 30 minutes at room temperature.
5. Decant the Zymogram Renaturing Buffer and replace with 1x Zymogram Developing Buffer (100 ml for one or two mini-gels). Equilibrate the gel for 30 minutes at room temperature with gentle agitation then replace with fresh 1x Zymogram Developing Buffer and incubate at 37℃ for at least four hours. Incubate overnight for maximum sensitivity. Incubation time can be reduced to as little as one hour for concentrated samples. The optimal result can be determined empirically by varying the sample load or incubation time.
6. Stain with Coomassie Blue R-250 for 30 minutes. For maximum contrast, use a stain concentration of 0.5% (w/v) instead of the usual concentration of 0.1%. Gels should be destained with an appropriate Coomassie R-250 destaining solution (Methanol : Acetic acid : Water (50 : 10 : 40). Areas of protease activity will appear as clear bands against a dark blue background where the protease has digested the substrate.
Sample Buffer (2X)
0.5 M Tris-HCl, pH 6.8 2.5 ml
Glycerol 2.0 ml
10% (w/v) SDS 4.0 ml
0.1% Bromophenol Blue 0.5 ml
Distilled Water to 10.0 ml
Running Buffer (10x) (1X)
Tris Base 29 g 2.9 g
Glycine 144 g 14.4 g
SDS 10 g 1.0 g
Distilled Water to 1 L to 1 L
pH=8.3
Renaturing Buffer (10x)
Triton X-100, 25% (v/v) in water
Developing Buffer
(10x) (1X)
Tris base 12.1 g 50mM
Tris-HCl 63.0 g 50mM
NaCl 117 g 0.2M
CaCl2 7.4 g 5 mM
Brij 35 0.2% 0.02%
Distilled Water to 1 L
