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| Section: Plant Lab Protocols |
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| Methodology for Amino Acids and Proteins |
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Polyacrylamide disc gel electrophoresis
Analysis of proteins in their native form is carried out in polyacrylamide buffer gel. The classical disc electrophoresis using cylindrical gels has been described by Davis in 1964. On such occasions as isoenzyme studies, protein purity checking etc., electrophoresis in buffer gels is an essential procedure. For instance, a purified protein may consist of four subunits. When this is analyzed in denaturing conditions on SDS gels this would show four separate bands even though the protein is pure. In such case as evidence of protein purity, the appearance of a single band on a gel could be shown by running a normal buffer gel.
Principle
The separation of native (non-denatured) proteins in this method is based on both the charge and size of the protein. Since the native proteins are large and exist in quaternary structure, a gel of low percentage of acrylamide is used.
Materials
This gel system consist of a 7.5% separating gel with a stacking gel. In disc electrophoresis, the gels are cast in long cylindrical glass tubes. Prior to pouring the gel solution, the bottom of the tube is sealed with dialysis membrane or parafilm.
Various solutions used are as follows:
A. Acrylamide Stock Solution (Separating Gel)
Acrylamide 28.0g
Bisacrylamide 0.74g
Water to 100mL
B. Acrylamide Stock Solution (Separating Gel)
Acrylamide 10.0g
Bisacrylamide 2.5g
Water to 100mL
(store A and B at 0-4°C in amber-colored bottles)
C. Tris- HCl (pH 8.9) Stock Solution
Tris 36.6g
1M HCl 4.8mL
TEMED 0.25mL
Water to 100mL
D. Tris- HCl (pH 6.7) Stock Solution
Tris 5.98g
1M HCl 49.0mL
TEMED 0.46mL
Water to 100mL
E. Ammonium Persulphate Solution
Dissolve 0.15g persulphate in 100mL distilled water. Prepare fresh each time.
F. Riboflavin Solution
0.4mg riboflavin in 10mL distilled water. Filter and use.
G. Electrode Buffer
Tris 0.6g
Glycine 2.9g
Water to 1L
Staining solution
0.1% Amido black 10B in 7% acetic acid.
Procedure
1.
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Separating Gel
Mix solution A:B:E:H2O in the ratio of 1:1:4:1
Degas the solution immediately and pour separating gel overlay with water and leave to set (30-60min).
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2.
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Stacking Gel
Mix solution B:D:F:H2O in the ratio of 2:1:1:4.
Degas immediately and pour the stacking gel. Leave to polymerize in bright light (use fluorescent light, desk lamp etc.)
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3.
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Load the sample extract (made to 15% sucrose, final) on the top of the gels.
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4.
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In the case of tube gels, initially apply 2mA/tube for 10min and then increase to 3mA/tube until the tracking dye travels to the bottom of the tube.
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5.
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Stain the proteins overnight using 0.1% Amido Black 10B in 7% acetic acid; destain in 7% acetic acid.
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Notes
1.
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The cylindrical glass tube (12 x 0.6cm ID) should be very clean as otherwise polymerization of the gel will be affected.
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2.
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Photopolymerization of the stacking gel using riboflavin as a catalyst is rather slow.
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3.
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To remove the cylindrical gel out of the tube after electrophoresis carefully inject water through a blunt-end needle between the glass tube and the gel.
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4.
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For analyzing a large number of samples, the slab gel system is made instead of cylindrical gels using the above solution and run essentially as described for SDS gels.
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References
1. Davis, B J (1964) Ann N Y Acad Sci. 121 404.
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