Gelatin zymography can be used for (1) the determination of gelatinase A (MMP-2) and gelatinase B (MMP-9), (2) the determination of inhibitors of gelatinases, and (3) the localization of gelatinases in cells and tissues.
Operation method
gelatin zymography
Principle
The extracellular matrix (ECM) is an important internal environment for cell survival, containing not only collagen, glycoproteins, proteoglycans, and other components, but also a large number of proteases, cytokines, and adhesion molecules.The ECM, and in particular the basement membrane therein, is a physiological barrier that must be overcome during tumor metastasis. Matrix metalloproteinases (MMPs) can degrade the extracellular matrix, helping tumor cells to overcome this barrier and providing space for cell proliferation.MMP2 and MMP9 are matrix metalloproteinases that mainly degrade type IV collagen and gelatin on the basement membrane, which are particularly closely related to the infiltration and metastasis of malignant tumors, and the activities of MMP2 and MMP9 can be detected by protein electrophoresis.
Materials and Instruments
Cell Homogenates Tissue Extracts Move I. Preparation of main reagents 1. Equilibrium buffer: 50 mmol/L Tris (pH 7.5), 0.5 mol/L NaCl, 10 mmol/L CaCl2, 0.01% Tween 20, 5 mmol/L o-diazophene. Equilibration buffer: 50 mmol/L Tris (pH 7.5), 0.5 mol/L NaCl, 10 mmol/L CaCl2, 0.01% Tween 20, 5 mmol/L o-diazafil. 2. 2. Wash buffer 1: 50 mol/L Tris (pH 7.5), 0.5 mmoL/L NaCl, 10 mmol/L CaCl2, 0.05% Tween 20, 5 mmol/L o-diazophene. 3. Wash buffer 2: 50 mmol/L Tris (pH 7.5), 10 mmoL/L CaCl2, 0.01% Tween 20, 5 mmol/L o-diazafil. 4. 8% separation gel (containing 0.1% gelatin): 30% acrylamide 2.7 ml, ddH20 4.5 ml, 1.5 mol／L Tris (pH 8.8) 2.5 ml, 10% SDS 100 μl, 10% ammonium persulfate (APS) 100 μl, TEMED 6 μl. 5. 5. Concentrated gel: ddH20 2.1 ml, 30% acrylamide 0.5 ml, 1 mol/L Tris-HCI (pH 6.8) 0.38 ml, 10% SDS 30 μl, 10% ammonium persulfate 30 μl, TEMED 3 μl. 6. 5×Tris-glycine electrophoresis buffer (pH 8.3): 0.125 mol/L Tris-HCl, 1.25 mol/L glycine, 0.5% SDS. 7. 5×sampling buffer: 30% 1 mol／L Tris-HCl (pH6.8), 25% glycerol, 0.05% bromophenol blue. 8. Elution solution: 2.5% TritonX-100 (deionized water). 9. Incubation solution: 50 mmoL/L Tris-HCl, 10 mmol/L CaCl2, 1% TritonX-100. pH 7.5, deionized water. 10. 10. Staining solution: 0.25% Kaomas Brilliant Blue R-250, 45% methanol, 10% acetic acid. 11. 11. Decolorizing solution: 30% methanol, 10% acetic acid. Experimental methods 1. Collect the culture medium of cancer cells in logarithmic stage of growth. 2. 2. Mix 500 μl of culture medium, 45 μl of gelatin agarose particles (mixed well before use) and 45 μl of equilibration buffer, then add them into the miniature rotary column, and equilibrate on the rotary mixer for 30 min. 3. 3. Wash the agarose pellet with washing buffer 1, 100 μl each time for 3 times, and centrifuge at 7000 r/min to remove the washing buffer 1. 4. 4. Wash the agarose pellet with 100 μl of Wash Buffer 2 for 1 time and centrifuge at 7000 r/min to remove Wash Buffer 2. 5. Add 40 μl of 2x Sampling Buffer to the micro-rotation column and centrifuge at 7000 r/min. The centrifuged upsampling buffer was reintroduced into the micro-spin column and centrifuged at 12000r/min for 10s. 6. Electrophoresis was carried out using 8% separator gel for 2.5h at a constant voltage of 130V. 7. Wash the protein gel with elution solution on a shaker 2 times for 30 min each to remove SDS from the gel. 8. Incubate the protein gel at 37℃ for 24h to make MMP decompose gelatin. 9. Stain on shaker for 1h and decolorize until the bands are clear. 10. Protein imaging: take pictures with a gel imager. Caveat 1. The preparation of polyacrylamide should be careful to eliminate air bubbles. 2. the activity of gelatin enzyme spectrum is affected by calcium ions, zinc ions and pH value and other factors, so the buffer preparation should be strictly accurate, try to use ultra-pure water, the incubation temperature should be well controlled. 3. In order to prevent the denaturation and inactivation of the enzyme in the samples, it is forbidden to boil the protein samples, and the sampling buffer should not contain β-mercaptoethanol or DTT. 4. The pH of the incubation solution should be 7.5-7. 6， and the reagent Triton will flocculate if it is left for too long, so try to use freshly configured solution in the experiments. 5. Incubate at 37℃ do not incubate atCO2incubator, because it will change the pH value of the incubation solution, in the ordinary incubator can be. 6. Gelatin should be stored at 4°C and used within 1 week after preparation. Common Problems Source Methods for the Study of In Vitro Models of Oncology Therapeutic Drugs. For more product details, please visit Aladdin Scientific website.
Polyacrylamide Gel Gelatin Gelatin Concentrate Tris-Glycine Buffer Triton X-100 Ammonium Persulfate Methanol Acetic Acid Thomas Blue Protein Sampling Buffer
Electrophoresis bad pH meter Gel Imager Centrifuge Electrophoresis Instrument Cell Culture Flasks Inverted Microscope Ultra-clean Bench Autoclave Electronic Balance Liquid Nitrogen Tank Micro Rotary Column Rotary Mixer