Western blotting Protein gel electrophoresis technical handbook separate transfer detect 2 Comprehensive solutions designed to drive your success Protein gel electrophoresis is a simple way to separate proteins prior to downstream detection or analysis, and is a critical step in most workflows that isolate, identify, and characterize proteins. We offer a complete array of products to support rapid, reliable protein electrophoresis for a variety of applications, whether it is the first or last step in your workflow. Our portfolio of high-quality protein electrophoresis products unites gels, stains, molecular weight markers, running buffers, and blotting products for your experiments. For a complete listing of all available products and more, visit thermofisher.com/separate For orderingordering informationinformation referrefer to pagepage XX.XX ForForqr quickquickrk referencereferencee on the protocolprotocol pleasepleasere referrefertr totopo pagepage XX. Prepare samples Choose the electrophoresis Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain 3 Contents Electrophoresis overview 4 Select precast gel Gel selection guide 8 Gels 10 Prepare samples and select buffers Sample prep kits 26 Buffers and reagents 28 Buffers and reagents selection guide 29 Select the standard Protein ladders 34 Protein standards selection guide 36 Choose the electrophoresis chamber system and power supply Electrophoresis chamber systems 50 Electrophoresis chamber system selection guide 51 Power supplies 58 Run the gel Gel run conditions 59 Troubleshooting tips 60 Stain the gel Protein stains 62 Protein stains selection guides 63, 67, 69, 70 Electrophoretic staining technology 71 Post stain Transfer and detection 74 Appendix Protocol quick reference 76 Ordering information 81 Sample preparation and Electrophoresis chamber Electrophoresis Precast protein gels Protein standards Protein gel stains electrophoresis buffers systems and power supplies run conditions 4 Electrophoresis Electrophoresis is defined as the Support matrix transport of charged molecules Two types of support matrices are commonly used in electrophoresis—polyacrylamide and agarose. The support through a solvent by an electric matrices act as porous media and behave like a molecular sieve. Separation of molecules is dependent upon the gel pore size of field. Electrophoresis is a simple, the support matrix used. Agarose has a large pore size and is ideal for separating macromolecules such as nucleic acids and protein rapid, and sensitive analytical complexes. Polyacrylamide has a smaller pore size and is ideal for separating most proteins and smaller nucleic acids. tool for separating proteins and Polyacrylamide gel electrophoresis nucleic acids. Any charged ion or (PAGE) molecule will migrate when placed Polyacrylamide gels are generated by the polymerization of in an electric field. Most biological acrylamide monomers. These monomers are crosslinked into long chains by the addition of bifunctional compounds such as molecules carry a net charge at any N,N,-methylenebisacrylamide (bis), which react with the free functional groups of the chain termini. The concentration of pH other than at their isoelectric acrylamide and bisacrylamide determines the pore size of the gel. The higher the acrylamide concentration, the smaller the pore size, point and will migrate at a rate resulting in resolution of lower molecular weight molecules and vice versa. proportional to their charge density. PAGE allows one to separate proteins for different applications based on: The mobility of a biological molecule • The acrylamide matrix through an electric field will depend • Buffer systems on the following factors: • Electrophoresis conditions • Field strength • Net charge on the molecule • Size and shape of the molecule • Ionic strength • Properties of the matrix through which the molecules migrate (e.g., viscosity, pore size) Mini Gel Tank Prepare samples Choose the electrophoresis Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain 5 The acrylamide matrix Electrophoresis conditions Linear vs. gradient gels The separation of molecules is dependent on the electrophoresis conditions. Electrophoresis can be performed under the Gels that have a single acrylamide percentage are referred to as following conditions: linear gels, and those with a range are referred to as gradient gels. The advantage of using a gradient gel is that it allows the separation of a broader range of proteins than a linear gel. Denaturing conditions Electrophoresis is performed under denaturing conditions using Continuous vs. discontinuous gels an anionic detergent such as sodium dodecylsulfate (SDS). SDS denatures and unfolds the protein by wrapping around the Researchers occasionally refer to gels as continuous or hydrophobic portions. SDS binds at a ratio of ~1.4 g SDS per discontinuous. A continuous gel is a gel that has been formed gram of protein. The resultant SDS-protein complexes are highly from a single acrylamide solution in the entire gel cassette. negatively charged and are resolved in the gel based on their size. A discontinuous gel is formed from two acrylamide solutions, a small, low-percentage stacking gel where the protein wells reside, and a larger portion of gel that separates the proteins. In the Nondenaturing (native) conditions traditional Tris-glycine protein gel system, the proteins are stacked Electrophoresis is performed under nondenaturing (native) in the stacking gel between the highly mobile leading chloride ions conditions using buffer systems that maintain the native protein (in the gel buffer) and the slower, trailing glycine ions (in the running conformation, subunit interaction, and biological activity. During buffer). The reason for using the stacking gel is to improve the native electrophoresis, proteins are separated based on their resolution of the bands in the gel. These stacked protein bands charge to mass ratios. undergo sieving once they reach the separating gel. Reducing conditions Mini vs. midi protein gels Electrophoresis is performed under reducing conditions using β Commercial gels are available in two size formats, minigels and reducing agents such as dithiothreitol (DTT), -mercaptoethanol β midigels. Both gels have similar run lengths, but midigels are wider ( -ME) or tris(2-carboxyethyl)phosphine (TCEP). than minigels, allowing midigels to have more wells or larger wells. The reducing agents completely unfold the denatured proteins The additional wells in the midigels permit more samples or large into their subunits by cleaving the disulfide bonds between sample volumes to be loaded onto one gel. cysteine residues. Buffer systems Electrophoresis is performed using continuous or discontinuous buffer systems. A continuous buffer system utilizes only one buffer in the gel and running buffer. A discontinuous buffer system utilizes a different gel buffer and running buffer1. This system may also use two gel layers of different pore sizes and different buffer composition (the stacking and separating gel). Electrophoresis using a discontinuous buffer system results in concentration of the Did you know? sample and higher resolution. Arne Tiselius won the Nobel Prize in Chemistry Reference for electrophoretic analysis 1. Ornstein L (1964) Disc electrophoresis. 1. Background and theory. Ann N Y Acad Sci 121:321-349. of serum proteins in 1948. Sample preparation and Electrophoresis chamber Electrophoresis Precast protein gels Protein standards Protein gel stains electrophoresis buffers systems and power supplies run conditions 6 With the Tris-acetate system (Figure 3), three ions are Comparison of primarily involved: • Acetate (–), the leading ion from the gel buffer discontinuous buffer • Tricine (–), the trailing ion from the running buffer systems • Tris (+), the common ion (in both gel and running buffer) This system also operates at a significantly lower pH than the Tris- SDS-PAGE utilizes a discontinuous buffer system to concentrate glycine system, resulting in less gel-induced protein modifications. or “stack” samples into a very sharp zone in the stacking gel at the beginning of the run. In a discontinuous buffer system, the primary The diagrams below (Figures 1, 2, and 3) summarize the migration anion in the gel is different (or discontinuous) from the primary differences in the stacking gel of each system. anion in the running buffer. Both the Invitrogen™ NuPAGE™ systems (Bis-Tris and Tris-acetate gels) and the Laemmli (Tris-glycine) system are examples of discontinuous buffer systems and work in a similar fashion. However, the NuPAGE system operates at a lower pH as a result of the proprietary ions that are in the system. GLYCINE Figure 1. The Tris-glycine gel system. (Trailing Ion) • Gel buffer ions are Tris and chloride PROTEIN/SDS COMPLEX (pH 8.7) In a Tris-glycine system (Figure 1), three ions are primarily involved: (Stacked Proteins) • Running buffer ions are Tris, glycine, CHLORIDE • Chloride (–), supplied by the gel buffer, serves as the leading (Leading Ion) and SDS (pH 8.3) ion because it has the highest attraction to the anode relative to • Gel operating pH is 9.5 PROGRESSION OF RUN other anions in the system. Common Ion is Tris, present in the gel and running buffers • Glycine (–), the primary anion provided by the running buffer, serves as the trailing ion, because it is only partially negatively charged and remains behind the