& Data Analysis
Protein gel analysis
Keeping a lab notebook
Writing research papers
Dimensions & units
Using figures (graphs)
Examples of graphs
Principles of microscopy
Solutions & dilutions
Fractionation & centrifugation
Radioisotopes and detection
Guide to the study
Lab part 1
Lab part 2
Lab part 3
Introduction to SDS-PAGE
This material is accompanied by a presentation on protein structure and principles behind denaturing samples and discontinuous gel electrophoresis.
The separation of macromolecules in an electric field is called electrophoresis. A very common method for separating proteins by electrophoresis uses a discontinuous polyacrylamide gel as a support medium and sodium dodecyl sulfate (SDS) to denature the proteins. The method is called sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The most commonly used system is also called the Laemmli method after U.K. Laemmli, who was the first to publish a paper employing SDS-PAGE in a scientific study.
SDS (also called lauryl sulfate) is an anionic detergent, meaning that when dissolved its molecules have a net negative charge within a wide pH range. A polypeptide chain binds amounts of SDS in proportion to its relative molecuar mass. The negative charges on SDS destroy most of the complex structure of proteins, and are strongly attracted toward an anode (positively-charged electrode) in an electric field.
Polyacrylamide gels restrain larger molecules from migrating as fast as smaller molecules. Because the charge-to-mass ratio is nearly the same among SDS-denatured polypeptides, the final separation of proteins is dependent almost entirely on the differences in relative molecular mass of polypeptides. In a gel of uniform density the relative migration distance of a protein (Rf, the f as a subscript) is negatively proportional to the log of its mass. If proteins of known mass are run simultaneously with the unknowns, the relationship between Rf and mass can be plotted, and the masses of unknown proteins estimated.
Protein separation by SDS-PAGE can be used to estimate relative molecular mass, to determine the relative abundance of major proteins in a sample, and to determine the distribution of proteins among fractions. The purity of protein samples can be assessed and the progress of a fractionation or purification procedure can be followed. Different staining methods can be used to detect rare proteins and to learn something about their biochemical properties. Specialized techniques such as Western blotting, two-dimensional electrophoresis, and peptide mapping can be used to detect extremely scarce gene products, to find similarities among them, and to detect and separate isoenzymes of proteins.
Molecular mass versus molecular weight
Molecular mass (symbol m) is expressed in Daltons (Da). One Dalton is defined as 1/12 the mass of carbon 12. Most macromolecules are large enough to use the kiloDalton (kDa) to describe molecular mass. Molecular weight is not the same as molecular mass. It is also known as relative molecular mass (symbol Mr, where r is a subscript). Molecular weight is defined as the ratio of the mass of a macromolecule to 1/12 the mass of a carbon 12 atom. It is a dimensionless quantity.
When the literature gives a mass in Da or kDa it refers to molecular mass. It is incorrect to express molecular weight (relative molecular mass) in Daltons. Nevertheless you will find the term molecular weight used with Daltons or kiloDaltons in some literature, often using the abbreviation MW for molecular weight.
Polyacrylamide gels for SDS-PAGE
Many systems for protein electrophoresis have been developed, and apparatus used for SDS-PAGE varies widely. The methodology used on these pages employs the Laemmli method. Reference to the Laemmli method in a materials and methods section eliminates the need to describe the buffers, casting of gels, apparatus, etc. Unless the paper employs some modification to the method, the only details of SDS-PAGE that should be reported in a methods section are percent total acrylamide (%T) in a gel, relative percentage and type of crosslinker (%C), and perhaps a reference to the gel dimensions. We use a "mini-gel" system, with 3 1/4" x 4" gel cassettes.
SDS-PAGE can be conducted on pre-cast gels, saving the trouble and hazard of working with acrylamide. The following description applies to shop-made casting and running apparatus that are much cheaper than commercially available equipment. In addition to cost effectiveness, an advantage of making one's own gels the first time is a deeper understanding of the process.
Regardless of the system, preparation requires casting two different layers of acrylamide between glass plates. The lower layer (separating, or resolving, gel) is responsible for actually separating polypeptides by size. The upper layer (stacking gel) includes the sample wells. It is designed to sweep up proteins in a sample between two moving boundaries so that they are compressed (stacked) into micrometer thin layers when they reach the separating gel.