Chapter 31 Protein Gel Staining Methods: An Introduction and Overview
Introduction
This chapter focuses on electrophoresis-based protein detection with emphasis on current methods for detection of total protein and detection of the most frequently encountered protein posttranslational modifications (PTMs), phosphorylation, and glycosylation. In the 20 years since the publication of first edition of this volume, denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) followed by protein staining has remained a fundamental laboratory procedure to determine the composition and characterization of protein-containing mixtures at all stages of a purification scheme. It is standard practice to display protein profiles from a sampling of all purification steps—cell lysate to the final product—to evaluate polypeptide composition, purity, and quantitation. Thus a sample from each step should reveal a progressively simpler polypeptide profile showing a greater proportion of the target protein.
The previously summarized methods and principles of protein electrophoresis and detection remain relevant (Dunbar et al., 1990, Garfin, 1990a, Garfin, 1990b, Merril, 1990).
The widespread use of SDS–PAGE protein analytical methods has driven the commercial development and common use of standardized electrophoresis apparatus and reagents, precast multiwell 1D and 2D slab gels (minigels; ca. 8 × 8 cm, × 1.0 mm thick), and ready-to-use staining formulations or kits for simplified, rapid staining. Additionally, proteomics-oriented electrophoresis techniques have emerged, directed toward analysis of complex protein mixtures by large format 2-D gel electrophoresis followed by mass spectrometry (MS) of excised protein spots. The utility of gel-based methodology in proteome analysis has spurred further commercial development of extant colorimetric stains for total protein and development of new fluorescent stains and modalities for total protein and for protein PTMs, driving the coevolution of instrumentation for sensitive detection, easy documentation, and accurate quantitation of both fluorescent and colorimetric signals. A recent compendium of proteomics protocols describes methods and applications for protein detection in 2-D and 1-D gels and subsequent analytical methods (Walker, 2005). Recent reviews of protein detection in gels for proteomics summarize the history and scope of gel staining and detection methods (Miller et al., 2006, Patton, 2000, Patton, 2002, Smejkal, 2004). Clearly, there is convergence in the considerations and methods for protein identification in gels for proteomic analysis and protein purification.
Zymography—in-gel enzymatic activity analysis—is a complementary, gel-based protein characterization methodology beyond the scope of this chapter. A comprehensive handbook of zymographic protocols has recently been published (Manchenko, 2003). A related, emerging field—activity-based proteomics—utilizes small molecule probes to covalently link reporter moieties to catalytic sites of specific classes of active enzymes in complex mixtures such as lysates or whole cells. This increasingly popular approach can be used to in-gel assays to identify enzymes and to track their purification (Jessani and Cravatt, 2004, Paulick and Bogyo, 2008).
A detection method that accurately evaluates the total protein profile is usually sufficient for a general purification protocol. Protein detection in SDS–PAGE can be done by labeling samples—by covalent modification with a fluorescent dye—prior to running gels or, more commonly, by noncovalent postelectrophoresis staining with organic dyes or by metal deposition techniques. Posttranslational modifications can be measured by specialized staining formulations and protocols that target the modified moieties in a background of total protein. PTM detection methods always require a control lane displaying proteins known to contain and others known to be devoid of the targeted PTM. If selective PTM detection utilizes fluorescence, bear in mind that intrinsic protein fluorescence may be a confounding factor, especially if ultraviolet (UV) light-excited, blue or green light-emitting fluorophores are used. Therefore, an additional control—chemical or enzymatic treatment of a sample—to remove the postulated PTM may be necessary.
Section snippets
General Considerations
Common features of all gel staining protocols are grounded in good laboratory practices: cleanliness, careful manipulations, and attention to detail. Postelectrophoresis gel staining is generally accomplished in covered polycarbonate or polypropylene dishes. Accumulation of residual stain may compromise results, particularly with fluorescent stains. Dishes should be cleaned with 70–100% ethanol or methanol followed by a water rinse. The gels are incubated on a rocking platform or an orbital
Instrumentation: Detection and Documentation
Obviously, detection by visual inspection has been the basis of protein gel staining; documentation has been accomplished by photography of stained gels or by drying stained gels and pasting them into a laboratory notebook. Unless quantitative data are required, most purification detection and documentation requirements now can be met with relatively low-cost flatbed scanners or with camera mounted visible or UV light box stations. In the past two decades, instrumentation for detection and
Colorimetric total protein stains
Simplicity of detection by visual inspection, relative ease of use, and widespread familiarity among the user base continue to ensure that staining with Coomassie Brilliant Blue (CBB) is the most commonly used total protein gel stain, and silver staining continues to be the alternative colorimetric method, for increased detection sensitivity over Coomassie staining. If mass spectrometry of excised protein is desired, staining methods that are do not introduce covalent protein modifications are
Phosphoprotein Detection
The importance of reversible protein phosphorylation at selected amino acid residues as a fundamental cell signaling mechanism is beyond dispute. Current phosphoprotein stains are proprietary formulations with phosphate-binding moieties covalently attached to fluorophores. The mode of detection is selective binding to phosphorylated amino acids, but with no fluorescent enhancement. Many soluble fluorescent compounds can be, to some degree, total protein stains. A selective phosphoprotein
General glycoprotein detection
Glycosylation is the most frequent protein posttranslational modification in eukaryotes. Oligosaccharides are usually linked to asparagine side chains (N-linked glycosylation) or to serine and threonine hydroxyl side chains (O-linked glycosylation). Postelectrophoretic glycoprotein staining in gels generally utilizes periodate oxidation of vicinal glycol residues followed by hydrazide conjugation by a Schiff's base mechanism.
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