- Sodium Dodecylsulfate Polyacrylamide Gelelectrophoresis
- analysis of purity and molecular mass of proteins
- separation according to their size only
Location:
Arnimallee 22, E030Altensteinstraße 23 a, 115
Responsible person:
Michael Tully, Arnimallee 22, E030, michael.tully@fu-berlin.de
Stefanie Wedepohl, Arnimallee 22, E001, stefanie.wedepohl@fu-berlin.desee OpenIRIS
Assay principle
SDS-PAGE is a method to separate and analyze proteins in the range of 5-250 kDa mainly by size. This is achieved by using SDS, a surfactant that binds to proteins and thereby masks their individual charge, resulting in equally distributed negative charge on the protein surface. The protein's secondary structure can be further eliminated by heat denaturation and/or reduction of the disulfide bonds. Proteins are loaded onto a polyacrylamide gel matrix and exposed to an electric field, where they migrate due to their charges. Smaller proteins migrate in the gel matrix faster than larger proteins. Because of the uniform negative charge mediated by SDS, separation is therefore dependent only on the size of the proteins and the gel mesh.
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After destaining, the bands can be documented via the Gel-Doc system or transfered to membranes for western blot analysis.
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2 separating gels | 7,5 % | 10 % | 13 12 % | ||
1,5 M Tris HCl pH 8,8 | 2,5 ml | 2,5 ml | 2,5 ml | ||
Rotiphorese 30 (Roth) | 2,5 ml2 | 3,68 33 ml3,5 | 4 ml | ||
ddH2O | 4,79 ml | 3,32 96 ml | 23,5 28 ml | ||
10 % SDS | 0,08 ml | 0,08 ml | 100 µl | 100 µl | 100 µl0,08 ml |
10% APS | 40 100 µl40 | 100 µl | 40 100 µl | ||
TEMED | 6 13,4 µl6 | 13,4 µl | 6 13,4 µl |
- start the polymerization by addition of TEMED and immediately pour the solution between the plates until the marking.
- add some isopropanol to the surface of the gel to break bubbles
- wait until polymerized, then remove the isopropanol, rinse with water and dry with filter paper
- prepare mixture for stacking gels:
2 stacking gels | |
0,5 M Tris HCl pH 6,8 | 1,25 ml |
Rotiphorese 30 | 0,67 8 ml |
ddH2O | 2,33 35 ml |
10% SDS | 0,04 ml50 µl |
10% APS | 20 50 µl |
TEMED | 2 10 µl |
- add TEMED when ready for polymerization
- pour mixture on top of the separating gel
- immediately insert comb
- make sure all pockets are closed tight, i.e. no air bubbles trapped
- wait until polymerized (about 20 minutes)
- use immediately or store under conditions where it doesn't dry out (in a plastic bag in the fridge)
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View file name BioRadMiniProteanII.pdf height 250
Link to gel documentation system
