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Gelatin Zymography
This technique involves the electrophoresis of secreted protease enzymes through discontinuous polyacrylamide gels containing enzyme substrate (either type III gelatin or B-casein). After electrophoresis, removal of SDS from the gel by washing in 2.5% Triton X-100 solution allows enzymes to renature and degrade the protein substrate. Staining of the gel with commassie allows the bands of proteolytic activity to detected as clear bands of lysis against a blue background.
Gelatinase A and B enzymes were analysed on gelatin containing gels ( 7.5% polyacrylamide gel containing 2mg/ml gelatin) and stromelysin enzymes on B-casein containing gels (11% polyacrylamide gel containing 2mg/ml casein). Casein substrate was incorporated into gels by slowly dissolving 20mg of B-casein in 2.5 ml of Tris-HCl, pH 8.8. Insoluble material was removed by centrifugation. After addition of substrate to the resolving gel mixture, gels were cast between the glass plates of a Bio Rad Mini Protean II electrophoresis apparatus.
Conditioned medium samples for analysis by casein zymography were concentrated 10 - 40 fold using Amicon Centriprep 30 concentrators.
Samples were centrifuged in the concentrator at 6000 rpm in a J10 rotor for 1 - 1.5 hr. Samples were then mixed with an equal volume of 2 x non reducing samples buffer and 15 - 20 ul loaded per well. In addition to conditioned medium samples, 10ul of denatured molecular weight markers, consisting of B-galactosidase (205kD), phosphorylase b (116kD), bovine albumin (66kD), egg albumin (45kD) and carbonic anhydrase (29kD), were also electrophoresed on each gel.
Gels were electrophoresed at 90V at 4oC in 1x running buffer untill the bromophenol blue marker dye reached the bottom of the gel. After electrophoresis SDS was removed from the gel by washing 3 x 10 min in 2.5% Triton X-100 solution. This allows the MMPs to renature and digest the surrounding substrate when incubated overnight at 37oC in zymogram incubation buffer. After incubation, the gel is stained with a solution of 0.25% Coomassie blue R250, 40% methanol and 10% acetic acid for 2 hr at room temperature and destained with 40% methanol, 10% acetic acid untill the bands of lysis become clear.
Preparation of Gels:
Reagent
7.5% Gelatin Gel
4% Stacking Gel
ddH2O
3.85
6.10
20mg/ml gelatin
substrate
1.00
-
30% Acrylamide
2.50
1.30
1.5M Tris-HCl, pH 8.8
2.50
-
0.5M Tris-HCl, pH 6.8
-
2.50
10% SDS
0.10
0.10
10% APS
0.10
0.10
TEMED
0.006
0.01
Preparation of Solutions/Buffers:
Solution/Buffer
Contents
Recipe
Storage
Incubation Buffer
50mM Tris-HCl, pH 7.6,
10mM CaCl2.2H2O, 50mM NaCl, 0.05% Brij35
For 1 L
6.06g Tris, 1.47g CaCl2, 2.92g NaCl, 0.5g Brij35.
Make up to 800ml with HPLC water, pH to 7.6 with HCl. add water to final volume of 1L
4oC
5 x Running Buffer
125mM Tris-HCl, pH 8.3, 1.23M Glycine, 0.5% SDS
For 1L
15.1g Tris, 94g Glycine, 5g SDS. Make up to 800ml with HPLC water, pH to 8.3. Add water to final volume of 1L.
4oC
2 x Non-reducing buffer
for 8ml, mix 2.8ml dH2O; 1ml 0.5M Tris-HCl, pH 6.8; 0.8ml glycerol; 3.2ml 10% (w/v) SDS; 0.2ml 0.2% bromophenol blue.
Room temperature
30% Acrylamide solution
29.2% Acrylamide; 0.8% N’N’-Bis-methylene acrylamide
Dark 4oC
Step-by step.
1 Clean electrophoresis apparatus in warm water + detergent and rinse thoroughly. Clean glass plates in 70% ethanol.
2 Set up plates. Large plate, then two spacers and small plate on top. Assemble plates into clamp and gently tighten screws. Do not over tighten, as this will crack the plates.
3 Put clamp assembly onto the rubber gasket stand ready to pour in gel mix.
4 Mix the appropriate resolving gel mix in a universal tube and pipette between the plates avoiding bubbles. Fill plates about 80% way up leaving space for the stacking gel and comb. Overlay with a small amount of saturated butanol to achieve a completely flat interface between resolving gel and stacking gel. Allow to set for about 10 minutes.
5 While resolving gel in setting prepare the stacking gel. 10mls is enough for two gels.
6 When the resolving gel in set pour off the excess water/butanol and wash between the plates with distilled water. Remove excess with whatmann paper.
7 Pour in stacking gel and inset comb avoiding bubbles. Allow to set for about 10 mins.
8 When stacking gel is set gently remove comb and assemble gels onto the electrode/gasket section of the gel apparatus. Fill central well with 1x running buffer and the bottom of the tank with 1x running buffer so that it reaches the bottom of the plates.
9 Prepare samples. i.e mix with equal volume of 2x non-reducing sample buffer or 1/5th vol of 5x sample buffer and pipette into wells using gel loading tips.
10 Put lid on tank and plug cables into power supply.
11 Run at about 90V at 4oC until the bromo-phenol blue reaches the bottom of the plates.
12 Disassemble the apparatus and gentle wash the gel into a plastic dish using 2.5% Triton-X-100. Wash gel 2x15min in 2.5% Triton-X-100 to remove SDS from the gel.
13 Incubate gel in zymogram incubation buffer at 37oC overnight.
It should look something like this.. The background stains blue with coomassie stain as the gel contains gelatin. Where the gelatin is degraded white bands appear indicating the presence of gelatinases. The lower bands are gelatinase-A (MMP-2) which is about 72kD while the upper bands are gelatinase-B (MMP-9) which runs at about 95kD.
