Assignment: Performing Protein Electrophoresis

MCB 253 Protein Characterization: SDS-PAGE © Elizabeth A. Good University of Illinois at Urbana-Champaign S SDS-PAGE S Sodium Dodecyl Sulfate – Polyacrylamide Gel Electrophoresis S This protocol utilizes the properties of the detergent SDS to resolve proteins on a polyacrylamide gel by means of electrophoresis. SDS S SDS is a negatively charged detergent which binds to hydrophobic regions of protein molecules. S It causes molecules to unfold. S It releases individual proteins from their intermolecular and intramolecular associations. S It conveys a net negative charge to all proteins treated with it. SDS and 2-Mercaptoethanol (BME) MBL Life science Mini-PROTEAN® Tetra Vertical Electrophoresis Cell and Power Supply Precast Gel S Percentage of gel: 10%, 12%, or 4-20% S Remove comb before running gel S Remove tape at the bottom before running gel Electrophoresis S An electric field facilitates the migration of proteins. S The top of the gel insert is negatively charged while the bottom of the gel insert is positively charged. S Negatively charged proteins migrate toward the positive pole. Protein Sample Preparation S Purified protein sample S Choose sample buffer S Dilutions- what concentration of protein per well? S Each gel has 10 wells.

You should run two MW and one lane for each of your five (#1-5) protein samples. S Each well holds 20ul. S You may run two gels in the same chamber, but they must be the same %. S If you choose to run one gel, then you must use a buffer dam to hold the place of what would be the second gel. Sample Buffers S 2x Sample buffer + SDS S 2x Sample buffer + SDS + BME Both sample buffers contain glycerol and bromophenol blue- why? Electrode Buffers S Electrode/Running buffer with SDS S Tris base + glycine + SDS Molecular Weight Standard Prestained Precision Plus Protein™ Kaleidoscope™ Standards #161-0375 Load samples and Run the Gel Sigma Visualization S Protein bands on a gel can be visualized by several methods: S Coomassie blue stain (1-5 mg of a purified protein) S Western blot and subsequent detection with antibodies and HRP- color development solution S Western is longer lasting. Analyzing the DataCreating a Molecular Weight Graph (Determine the MW of your protein) To determine the molecular weight of each your protein samples (#1-5), do the following: 1. Measure the distance migrated by each protein standard on the gel (from the bottom of the well to the center of each protein standard band). 2. On semi-log paper (provided for you in the Appendix of your lab manual) plot the MW against the distance migrated for each protein standard band. This will create your standard curve. 3. Measure the distance migrated for each band of your protein. 4.

Determine your protein’s weight by extrapolating the value from your standard curve. Why would your sample protein have more than one band in a well? Standard curve example: TA Help Sessions S Day: Friday S Time: 10am-1pm or 2-5pm S Location: Online in Zoom (Link posted on our course Moodle page) Upcoming Week 5 Activities Week 5: February 22-26, 2021: S SDS-Page Experiment S Data Analysis Group Work S SDS-PAGE gel images will be provided to you. Upcoming Assignment Deadlines Due on Friday, February 19, 2021: S Bradford Data Analysis Electrophoresis A Guide to Polyacrylamide Gel Electrophoresis and Detection BEGIN Electrophoresis Guide Table of Contents Part I: Theory and Product Selection 5 Chapter 1 Overview Buffer Systems and Gel Chemistries 5 31 Bis-Tris 31 How Protein Electrophoresis Works 6 Tris-Acetate 31 General Considerations and Workflow 6 Tris-Tricine 31 IEF 31 Products for Handcasting Gels Chapter 2 Protein Electrophoresis Methods and Instrumentation 9 Protein Electrophoresis Methods Polyacrylamide Gel Electrophoresis (PAGE) Discontinuous Native PAGE 32 AnyGel™ Stands 32 10 Multi-Casting Chambers 32 10 Gradient Formers 32 10 11 12 Other Types of PAGE Blue Native PAGE (BN-PAGE) 12 Zymogram PAGE 12 Isoelectric Focusing (IEF) 12 2-D Electrophoresis 13 13

TABLE OF CONTENTS Electrophoresis Cells 13 Power Supplies for PAGE Applications 15 Chapter 5 Performing Electrophoresis System Setup General Considerations 17 35 36 General Tips for Sample Preparation 52 52 Lysis (Cell Disruption) 52 Protein Solubilization 52 Preparation for PAGE 52 Human Cells 53 Suspension Cultured Cells 53 Monolayer Cultured Cells 53 Mammalian Tissue 54 Plant Leaves 54 Microbial Cultures 55 Protein Fractions from Chromatography 55 Sample Quantitation (RC DC™ Protein Assay) 56 36 Standard Assay Protocol (5 ml) 56 36 Microfuge Tube Assay Protocol (1.5 ml) 56 Joule Heating Other Factors Affecting Electrophoresis Selecting Power Supply Settings Separations Under Constant Voltage Separations Under Constant Current Separations Under Constant Power 36 57 37 Pour the Resolving Gel 58 37 Pour the Stacking Gel 58 37 Gradient Gels 37 Gel Disassembly and Storage 37 19 Chapter 6 Protein Detection and Analysis Detergents 20 Protein Stains Reducing Agents 20 Chaotropic Agents 21 Buffers and Salts 21 Common Solutions for Protein Solubilization 21 57 37 General Guidelines for Running Conditions 20 Handcasting Polyacrylamide Gels Single-Percentage Gels 19 39 Performing Electrophoresis General Protocols: SDS-PAGE Total Protein Staining 59 60 60 62 Bio-Safe™ Coomassie Stain 62 Oriole™ Fluorescent Gel Stain 62 40 Flamingo™ Fluorescent Gel Stain 62 Total Protein Stains 40 63 Specific Protein Stains 40 Dodeca™ High-Throughput Stainers 42 Silver Staining (Bio-Rad Silver Stain) Molecular Weight Estimation Buffer Formulations 63 64 42 Sample Preparation Buffers 64 Imaging Systems 42 Gel Casting Reagents 65 Imaging Software 43 Sample Buffers 65 Running Buffers 66 Buffer Components 66 Imaging Removal of Interfering Substances 21 Immunoprecipitation 22 Sample Quantitation (Protein Assays) 22 Molecular Weight (Size) Estimation 44 23 Quantitation 44 Total Protein Normalization 45 25 Sample Preparation 52 36 Protein Solubilization Chapter 4 Reagent Selection and Preparation Protocols 51 Useful Equations Cell Disruption Protein Assays

Part II: Methods Running Conditions Chapter 3 Sample Preparation for Electrophoresis 32 Premade Buffers and Reagents SDS-PAGE Electrophoresis Cells and Power Supplies Analysis Chapter 7 Downstream Applications 44 47 Part III: Troubleshooting 69 Sample Preparation 70 Gel Casting and Sample Loading 70 General Considerations 26 Western Blotting (Immunoblotting) 48 Electrophoresis 71 Protein Standards 26 Immunodetection 48 Total Protein Staining 72 Evaluation of Separation 73 49 Part IV: Appendices 77 49 Glossary 78 49 References and Related Reading 83 Ordering Information 86 Recombinant Standards Polyacrylamide Gels 26 PrecisionAb™ Validated Antibodies for Western Blotting 48 27 Immun-Star AP & HRP Secondary Antibody Conjugates 48 Fluorescent secondary antibodies for multiplex western blotting 49 Polymerization 27 Percentage 28 StarBright™ Blue 700 Secondary Antibodies Precast vs. Handcast 28 Format (Size and Comb Type) 2 29 Laemmli (Tris-HCl) 29 hFAB anti-Housekeeping antibodies Electroelution 3 Electrophoresis Guide Chapter 1: Overview Theory and Product Selection PART I TABLE OF CONTENTS Theory and Product Selection CHAPTER 1 Overview Protein electrophoresis is the movement of proteins within an electric field. Popular and widely used in research, it is most commonly used to separate proteins for the purposes of analysis and purification. This chapter provides a brief overview of the theory and workflow behind protein electrophoresis. 4 5 Electrophoresis Guide Chapter 1: Overview How Protein Electrophoresis Works The term electrophoresis refers to the movement of charged molecules in response to an electric field, resulting in their separation.

TABLE OF CONTENTS In an electric field, proteins move toward the electrode of opposite charge. The rate at which they move (migration rate, in units of cm2/Vsec) is governed by a complex relationship between the physical characteristics of both the electrophoresis system and the proteins. Factors affecting protein electrophoresis include the strength of the electric field, the temperature of the system, the pH, ion type, and concentration of the buffer as well as the size, shape, and charge of the proteins (Garfin 1990) (Figure 1.1). Proteins come in a wide range of sizes and shapes and have charges imparted to them by the dissociation constants of their constituent amino acids. As a result, proteins have characteristic migration rates that can be exploited for the purpose of separation. Protein electrophoresis can be performed in either liquid or gel-based media and can also be used to move proteins from one medium to another (for example, in blotting applications). Assignment: Performing Protein Electrophoresis

Over the last 50 years, electrophoresis techniques have evolved as refinements have been made to the buffer systems, instrumentation, and visualization techniques used. Protein electrophoresis can be used for a variety of applications such as purifying proteins, assessing protein purity (for example, at various stages during a chromatographic separation), gathering data on the regulation of protein expression, or determining protein size, isoelectric point (pI), and enzymatic activity. In fact, a significant number of techniques including gel electrophoresis, isoelectric focusing (IEF), electrophoretic transfer (blotting), and two-dimensional (2-D) electrophoresis can be grouped under the term “protein electrophoresis” (Rabilloud 2010). Though some information is provided about these methods in the following chapters, this guide focuses on the onedimensional separation of proteins in polyacrylamide gels, or polyacrylamide gel electrophoresis (PAGE). Power supply Theory and Product Selection Protein Electrophoresis Workflow Method Selection Electrodes Anode + – Cathode – – + + Consider the experimental goals in selecting the appropriate electrophoresis method. Instrumentation selection depends on the desired resolution and throughput. Sample Preparation – + Fig. 1.1. Movement of proteins during electrophoresis. Assignment: Performing Protein Electrophoresis

The protein sample may be prepared from a biological sample, or it may come from a step in a purification workflow. In either case, prepare the protein at a concentration and in a buffer suitable for electrophoresis. General Considerations and Workflow The electrophoresis workflow (Figure 1.2) involves the selection of the appropriate method, instrumentation, and reagents for the intended experimental goal. Once proteins are separated, they are available for a number of downstream applications, including enzymatic assays, further purification, transfer to a membrane for immunological detection (immunoblotting or western blotting), and elution and digestion for mass spectrometric analysis. Gel and Buffer Preparation Whether handcast or precast, the gel type used should suit the properties of the protein under investigation, the desired analysis technique, and overall goals of the experiment. Buffer selection depends on the gel type and type of electrophoresis performed. Performing Electrophoresis Gels are placed in the electrophoresis cell, buffer is added, and samples are loaded. Select running conditions that provide optimum resolution while maintaining the temperature of the system during separation. Related Literature Protein Blotting Guide, A Guide to Transfer and Detection, bulletin 2895 2-D Electrophoresis for Proteomics: A Methods and Product Manual, bulletin 2651 Protein Detection and Analysis Select a visualization technique that matches sensitivity requirements and available imaging equipment.

Fig. 1.2. Protein electrophoresis workflow. 6 7 Electrophoresis Guide Chapter 2: Protein Electrophoresis Methods and Instrumentation Theory and Product Selection TABLE OF CONTENTS CHAPTER 2 Protein Electrophoresis Methods and Instrumentation Consider the experimental goals in selecting the appropriate electrophoresis method; selection of instrumentation depends on the number and volume of samples, desired resolution, and throughput. This chapter describes the most common techniques and systems in use today. 8 9 Electrophoresis Guide Chapter 2: Protein Electrophoresis Methods and Instrumentation Protein Electrophoresis Methods Two types of buffer systems can be used: By choosing suitable separation matrices and corresponding buffer systems, a range of experimental objectives can be met using protein electrophoresis (Zewart and Harrington 1993). ■■ Polyacrylamide Gel Electrophoresis (PAGE) When electrophoresis is performed in acrylamide or agarose gels, the gel serves as a size-selective sieve during separation. As proteins move through a gel in response to an electric field, the gel’s pore structure allows smaller proteins to travel more rapidly than larger proteins (Figure 2.1). Assignment: Performing Protein Electrophoresis

For protein separation, virtually all methods use polyacrylamide as an anticonvective, sieving matrix covering a protein size range of 5–250 kD. Some less common applications such as immunoelectrophoresis and the separation of large proteins or protein complexes >300 kD rely on the larger pore sizes of agarose gels. ■■ ontinuous buffer systems use the same buffer C (at constant pH) in the gel, sample, and electrode reservoirs (McLellan 1982). Continuous systems are not common in protein separations; they are used mostly for nucleic acid analysis iscontinuous buffer systems use a gel separated D into two sections (a large-pore stacking gel on top of a small-pore resolving gel, Figure 2.2) and different buffers in the gels and electrode solutions (Wheeler et al. 2004) Direction of protein migration TABLE OF CONTENTS In gel electrophoresis, proteins do not all enter the gel matrix at the same time. Samples are loaded into wells, and the proteins that are closer to the gel enter the gel first. In continuous systems, the uniform separation matrix yields protein bands that are diffuse and poorly resolved. In discontinuous systems, on the other hand, proteins first migrate quickly through the In most PAGE applications, the gel is mounted between large-pore stacking gel and then are slowed as they two buffer chambers, and the only electrical path enter the small-pore resolving gel. As they slow down, between the two buffers is through the gel. Usually, the they stack on top of one another to form a tight band, gel has a vertical orientation, and the gel is cast with which improves resolution. Assignment: Performing Protein Electrophoresis

Discontinuous systems also a comb that generates wells in which the samples are use ions in the electrophoresis buffer that sandwich applied (Figure 2.1). Applying an electrical field across the proteins as they migrate through the gel, and this the buffer chambers forces the migration of protein into tightens the protein bands even more (Figure 2.2). and through the gel (Hames 1998). Discontinuous buffer systems provide higher resolution than continuous systems, and varying the buffers used in the sample, gel, and electrode chambers creates Cathode a variety of discontinuous buffer systems that can be used for a variety of applications. Discontinuous Native PAGE Well Buffer Larger (high MW) protein Protein band Smaller (low MW) protein Theory and Product Selection Anode Gel Fig. 2.1. Schematic of electrophoretic protein separation in a polyacrylamide gel. MW, molecular weight. The original discontinuous gel system was developed by Ornstein and Davis (Ornstein 1964, Davis 1964) for the separation of serum proteins in a manner that preserved native protein conformation, subunit interactions, and biological activity (Vavricka 2009). In such systems, proteins are prepared in nonreducing, nondenaturing sample buffer, and electrophoresis is also performed in the absence of denaturing and reducing agents. Data from native PAGE are difficult to interpret. Since the native charge-to-mass ratio of proteins is preserved, protein mobility is determined by a complex combination of factors. Since protein-protein interactions are retained during separation, some proteins may also separate as multisubunit complexes and move in unpredictable ways. Moreover, because native charge is preserved, proteins can migrate towards either electrode, depending on their charge. Assignment: Performing Protein Electrophoresis

The result is that native PAGE yields unpredictable separation patterns that are not suitable for molecular weight determination. Related Literature Gel Electrophoresis: Separation of Native Basic Proteins by Cathodic, Discontinuous Polyacrylamide Gel Electrophoresis, bulletin 2376 Stacking gel 4%T*, pH 6.8 Resolving gel 7.5%T to 15%T, pH 8.8 Fig. 2.2. Migration of proteins and buffer ions in a denaturing discontinuous PAGE system. A, Denatured sample proteins are loaded into the wells; B, Voltage is applied and the samples move into the gel. The chloride ions already present in the gel (leading ions) run faster than the SDS-bound proteins and form an ion front. The glycinate ions (trailing ions) flow in from the running buffer and form a front behind the proteins; C, A voltage gradient is created between the chloride and glycinate ions, which sandwich the proteins in between them; D, The proteins are stacked between the chloride and glycinate ion fronts. At the interface between the stacking and resolving gels, the percentage of acrylamide increases and the pore size decreases. Movement of the proteins into the resolving gel is met with increased resistance; E, The smaller pore size resolving gel begins to separate the proteins based on molecular weight only, since the charge-to-mass ratio is equal in all the proteins of the sample; F, The individual proteins are separated into band patterns ordered according to their molecular weights. * %T refers to the total monomer concentration of the gel (see Chapter 4 for more information). Nevertheless, native PAGE does allow separation of proteins in their active state and can resolve proteins of the same molecular weight. As a result, the rate at which SDS-bound protein migrates in a gel depends primarily on its size, enabling molecular weight estimation. SDS-PAGE The original Laemmli system incorporated SDS in the gels and buffers, but SDS is not required in the gel. Assignment: Performing Protein Electrophoresis

SDS in the sample buffer is sufficient to saturate proteins, and the SDS in the cathode buffer maintains the SDS saturation during electrophoresis. Precast gels (manufactured gels such as Bio-Rad’s Mini-PROTEAN® and Criterion™ Gels) do not include SDS and so can be used for either native or SDS-PAGE applications. A range of gel and buffer combinations can be used for native and SDS-PAGE, each with its own advantages (see Chapter 4 for more details). To overcome the limitations of native PAGE systems, Laemmli (1970) incorporated the detergent sodium dodecyl sulfate (SDS) into a discontinuous denaturing buffer system, creating what has become the most popular form of protein electrophoresis, SDS-PAGE. When proteins are separated in the presence of SDS and denaturing agents, they become fully denatured and dissociate from each other. In addition, SDS binds noncovalently to proteins in a manner that imparts: ■■ ■■ ■■ An overall negative charge on the proteins. Since SDS is negatively charged, it masks the intrinsic charge of the protein it binds A similar charge-to-mass ratio for all proteins in a mixture, since SDS binds at a consistent rate of 1.4 g of SDS per 1 g protein (a stoichiometry of about one SDS molecule per two amino acids) O– S O O� …