||||||||||| 11Protein Purification and Analysis PROTOCOLS & APPLICATIONS GUIDE APPLICATIONS & PROTOCOLS CONTENTS I. Introduction 1 X. Immunoassays: ELISA and Western Blot II. Choosing the Right Protein Purification Strategy 1 Analysis 19 A. Protein Purification 1 A. ELISA 19 B. Isolation of Protein Complexes 1 B. Western Blot Analysis 21 C. SDS-PAGE 21 III. Affinity Tags 2 D. Western Blot Analysis of Proteins from TNT® Cell-Free A. Polyhistidine 2 Expression Systems 23 B. Glutathione-S-Transferase 2 XI. Mass Spectrometry Analysis 24 C. HaloTag® Protein Tag 2 A. Trypsin 24 IV. Purification of Polyhistidine-Tagged Proteins 2 B. In-Gel Protein Digestion 24 A. Rapid Purification of Polyhistidine-Tagged Proteins Using C. In-Solution Protein Digestion 25 Magnetic Resins 2 D. Affinity Tag In Vitro Pull-Down Assay with Trypsin B. Medium- to Large-Scale Purification of Digestion and Protein Analysis 26 Polyhistidine-Tagged Proteins In Column or Batch Formats 5 E. Trypsin/Lys-C Mix, Mass Spec Grade 26 C. 96-Well Format For Purification of Polyhistidine-Tagged F. Alternative Proteases 26 Proteins 8 G. ProteaseMAX™ Surfactant, Trypsin Enhancer 30 V. Purification of GST-Tagged Proteins 8 H. In-Gel Digestion of Proteins Using Trypsin and ProteaseMAX™ Surfactant, Trypsin Enhancer 30 A. Rapid Purification of GST-Tagged Proteins Using Magnetic Resins 8 XII. Composition of Solutions 32 VI. Purification of HaloTag® Fusion Proteins 11 XIII.References 33 A. HaloTag® Protein Purification from Mammalian Cells 11 B. HaloTag® Protein Purification from E. coli 11 VII. Purification of Biotinylated Proteins 12 A. PinPoint™ Xa System and SoftLink™ Resin for Purification of Biotinylated Protein 12 VIII.Protein:Protein Interaction Analysis: In Vivo and In Vitro Methods 13 A. Mammalian Two-Hybrid Systems 13 B. HaloTag® Pull-Down Assays 15 C. In Vitro Pull-Down Assays 16 IX. Analysis of DNA:Protein Interactions 17 A. Gel Shift Assays 17 B. Chromatin Immunoprecipitation 18 Protocols & Applications Guide www.promega.com rev. 8/15 ||||||||||| 11Protein Purification and Analysis PROTOCOLS & APPLICATIONS GUIDE APPLICATIONS & PROTOCOLS I. Introduction purification strategies exist to address desired scale, throughput and downstream applications. The optimal Information about the regulation of protein expression, approach often must be determined empirically. protein modification, protein:protein interactions and protein function during different stages of cell development A. Protein Purification helps us understand the development and physiology of The best protein purification protocol depends not only on organisms. This complex analysis of protein function is a the protein being purified but also on many other factors major task facing scientists working in the proteomic field such as the cell used to express the recombinant protein today. Although the field of proteomics was first described (e.g., prokaryotic versus eukaryotic cells). Escherichia coli as the study of proteins encoded by the genome, the remains the first choice of many researchers for producing definition has now expanded to encompass all proteins recombinant proteins due to ease of use, rapid cell growth and protein functions, including protein isoforms and and low cost of culturing. Proteins expressed in E. coli can modifications, interactions, structure and high-order be purified in relatively high quantities, but these proteins, complexes (Tyers and Mann, 2003). especially eukaryotic proteins, may not exhibit proper A variety of proteomics techniques exist to characterize the protein activity or folding. Cultured mammalian cells might relationship between proteins and biological function (Zhu offer a better option for producing properly folded and et al. 2003). For example, scientists can apply protein functional mammalian proteins with appropriate pull-down assays, yeast two-hybrid systems (Fields and post-translational modifications (Geisse et al. 1996) . Song, 1989; Chien et al. 1991) and mammalian two-hybrid However, the low expression levels of recombinant proteins systems (Giniger et al. 1985; Lin et al. 1988) to study in cultured mammalian cells presents a challenge for their protein:protein interactions or use chromatin purification. As a result, attaining satisfactory yield and immunoprecipitation and gel shift assays to analyze purity depends on highly selective and efficient capture of protein:DNA interactions. In addition, protein-chip these proteins from the crude cell lysate. technology, mass spectrometry, traditional one- or To simplify purification, affinity purification tags can be two-dimensional gel electrophoresis and enzyme-linked fused to a recombinant protein of interest (Nilsson et al. immunosorbent assays (ELISA) are instrumental in protein 1997). Common fusion tags are polypeptides, small proteins identification. or enzymes added to the N- or C-terminus of a recombinant A fundamental step in studying individual proteins is protein. The biochemical features of different tags influence purification of the protein of interest. There are four basic the stability, solubility and expression of proteins to which steps of protein purification: 1) cell lysis, 2) protein binding they are attached (Stevens et al. 2001). Using expression to a matrix, 3) washing and 4) elution. Cell lysis can be vectors that include a fusion tag facilitates recombinant accomplished a number of ways, including nonenzymatic protein purification. methods (e.g., sonication or French press) or use of B. Isolation of Protein Complexes hydrolytic enzymes such as lysozyme or a detergent reagent A major objective of the proteomic field is the elucidation such as FastBreak™ Cell Lysis Reagent (Cat.# V8571). The of protein function and organization of the complex FastBreak™ Cell Lysis Reagent mediates in-medium lysis networks that are responsible for key cellular processes. of E. coli cells that express recombinant proteins without Analysis of protein:protein interaction can provide valuable interfering with downstream purification of tagged proteins insight into the cell signaling cascades involved in these (Stevens and Kobs, 2004) and requires only minor processes, and analysis of protein:nucleic acid interactions modifications for use with mammalian and insect cell lines often reveals important information about biological (Betz, 2004). Because purification of native proteins can be processes such as mRNA regulation, chromosomal challenging, affinity purification tags are often fused to a remodeling and transcription. For example, transcription recombinant protein of interest such that the tag is used to factors play an important role in regulating transcription capture or detect the protein. by binding to specific recognition sites on the chromosome, Here we provide guidelines on how to determine the best often at a gene’s promoter, and interacting with other protein purification strategy and include protocols based proteins in the nucleus. This regulation is required for cell on common affinity tags. We also describe popular tools viability, differentiation and growth (Mankan et al. 2009; and techniques for proteomics research. Gosh et al. 1998). II. Choosing the Right Protein Purification Strategy Analysis of protein:protein and protein:DNA interactions often requires straightforward methods for immobilizing Proteins are biological macromolecules that maintain the proteins on solid surfaces in proper orientations without structural and functional integrity of the cell, and many disrupting protein structure or function. This diseases are associated with protein malfunction. Protein immobilization must not interfere with the binding capacity purification is a fundamental step for analyzing individual and can be achieved through the use of affinity tags. proteins and protein complexes and identifying interactions Immobilization of proteins on chips is a popular approach with other proteins, DNA or RNA. A variety of protein to analyze protein:DNA and protein:protein interactions and identify components of protein complexes (Hall et al. Protocols & Applications Guide www.promega.com 11-1 rev. 8/15 ||||||||||| 11Protein Purification and Analysis PROTOCOLS & APPLICATIONS GUIDE APPLICATIONS & PROTOCOLS 2004; Hall et al. 2007; Hudson and Snyder, 2006). Functional C. HaloTag® Protein Tag protein microarrays normally contain full-length functional Often times protein fusion tags are used to aid expression proteins or protein domains bound to a solid surface. of suitable levels of soluble protein as well as purification. Fluorescently labeled DNA is used to probe the array and A unique protein tag, the HaloTag® protein, is engineered identify proteins that bind to the specific probe. Protein to enhance expression and solubility of recombinant microarrays provide a method for high-throughput proteins in E. coli. The HaloTag® protein tag is a 34kDa, identification of protein:DNA interactions. Immobilized monomeric protein tag modified from Rhodococcus proteins also can be used in protein pull-down assays to ® isolate protein binding partners in vivo (mammalian cells) rhodochrous dehalogenase. The HaloTag protein was or in vitro. Other downstream applications such as mass designed to bind rapidly and covalently to a unique spectrometry do not require protein immobilization to synthetic linker to achieve an irreversible attachment. The identify protein partners and individual components of synthetic linker may be attached to a variety of entities such protein complexes. See Section IX. as fluorescent dyes and