Determining Molecular Weight of Unknown Protein Samples

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. Determining Molecular Weight of Unknown Protein Samples

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 Protocol for performing protein electrophoresis: SDS-PAGE Purpose The aim of the experiment is to determine the molecular weight of five unknown protein samples. In order to so, a sodium dodecyl-sulfate polyacrylamide gel electrophoresis will need to be performed. Hypothesis It is hypothesised that smaller will migrate faster through the polyacrylamide gel, while larger proteins will migrate slower or lag behind. This will result in varying distances migrated by proteins, and distance of migration will be directly related to protein size. The proteins will be visible on gel and appear as blue bands since they will be stained with Coomassie blue. It will allow us to distinguish the distance each protein has migrated.

Background Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is the most popular form of protein electrophoresis and is commonly used to separate proteins based on their molecular weight (Nowakowski, Wobig, & Petering, 2014). SDS is an amphipathic surfactant, it is used in protein electrophoresis as a denaturant (Bio-rad Bulletin 6040 Rev A, n.d.). SDS binds to and unravels proteins from their tertiary conformities, rendering said proteins into an elongated rod-like form, masking its intrinsic charge and applying an overall negative charge to the protein (Bio-rad Bulletin 6040 Rev A, n.d.). In addition, SDS binds at a consistent rate (1.4g SDS per 1g protein) thus imparting a negative charge which is proportional to the molecular mass or polypeptide chain length (Nowakowski, Wobig, & Petering, 2014; Bio-rad Bulletin 6040 Rev A, n.d.).

This allows for protein molecular weight estimation since proteins will migrate, based on size alone, through the polyacrylamide gel. The polyacrylamide gel is a cross-linked matrix and acts as a sieve through which the proteins move in response to an electric field (Bio-rad Bulletin 6040 Rev A, n.d.). The rate of migration and separation is dependent on the acrylamide concentration, where a higher concentration (higher acrylamide percentage gel) results in slower migration and vice versa (Good, E., 2021). As in the aforementioned paragraph, proteins will be negatively charged and will therefore migrate toward the positively charges electrode – smaller proteins will migrate faster and will be found lower down in the gel and larger proteins slower and will be found higher up in the gel (that is, the higher up the protein in the gel, the larger the protein or molecular mass). Once the proteins are electrophoresed, Coomassie blue dye is generally used to stain the polyacrylamide gel for visualisation of the proteins. SDS-PAGE is used for several 2 purposes, namely, protein identification and quantification (western blotting), determining protein purity and estimating protein size (Nowakowski, Wobig, & Petering, 2014). Determining Molecular Weight of Unknown Protein Samples

The aim of this experiment is to determine the molecular weight of five unknown protein samples by SDS-PAGE. Size will be measured in relation to distance migrated by a molecular weight marker standard of known molecular weight. Materials and Methods Equipment ➢ ➢ ➢ ➢ ➢ ➢ ➢ ➢ Bio-rad Mini-PROTEAN® Tetra Vertical Electrophoresis Cell Bio-rad Mini-PROTEAN® Tetra Vertical Electrophoresis Power Supply Bio-rad Mini-PROTEAN® Tetra Vertical Electrophoresis Precast gel Filter paper Micropipettes (P100, P20, P10) Microfuge tubes PCR tubes Thermocycler Methods 1. Prepare the following buffers: ➢ Running buffer with SDS (10X): Mix 30.4g Tris base, 144.2g glycerine, 10g SDS and make up volume to 1L with distilled water. ➢ Running buffer with SDS (1X): Mix 100ml of 10X running buffer with 900ml with distilled water. ➢ Sample buffer with SDS (2X): Mix 0.98g Tris base, 10ml SDS (20%), 0.05g bromophenol blue, 7.98ml glycerol and make up volume to 50ml with distilled water. Adjust to pH6.8 with concentrated HCl. 3 ➢ Sample buffer with SDS and BME (2X): Mix 0.98g Tris base, 10ml SDS (20%), 0.05g bromophenol blue, 7.98ml glycerol, 5ml BME and make up volume to 50ml with distilled water. Adjust to pH6.8 with concentrated HCl. 2. Sample preparation ➢ Dilute samples as per Table 1 below Table 1. Dilution chart lane 1 Initial concentration (ug/uL) 1,5 Sample Volume (uL) 4 Buffer volume (uL) 20 Total final volume (uL) 24 Final Concentration (ug/uL) 0,25 Final amount of protein (ug) 6 lane 2 1,5 4 20 24 0,25 6 lane 3 1,5 4 20 24 0,25 6 lane 4 1,5 4 20 24 0,25 6 lane 5 1,5 4 20 24 0,25 6 Only 20uL will be loaded into each well. Therefore, 5ug of protein per well 3. Heat the diluted sample at 95°C for 5 min in a thermocycler. 4. Prepare gels and assemble the electrophoresis cell: ➢ Remove the comb from the top and tape from the bottom of the gels and assemble the electrophoresis cell as per manufacturer’s protocol ➢ Fill the upper (inner) buffer chamber of each core with 200 ml of 1× running buffer ➢ Fill the lower (outer) buffer chamber to the indicator mark for 2 gels (550 ml) ➢ Rinse each well using running buffer and a P20 pipette 5. Load the gel using a P20 pipette as indicated in Table below 4 Table 2: Samples loaded into SDS-PAGE Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 Lane 7 Lane 8 Lane 9 Lane 10 Lane11 Lane 12 blank blank MWM NC Protein 1 Protein 2 Protein 3 Protein 4 Protein 5 MWM blank blank (10uL) Negative control (20uL distilled water) (20uL of dilution as per Table 1) (20uL of dilution as per Table 1) (20uL of dilution as per Table 1) (20uL of dilution as per Table 1) (20uL of dilution as per Table 1) (10uL) Molecular weight marker (MWM) used: Prestained Precision Plus Protein™ Kaleidoscope™ Standards #1610375 6. Connect to the power supply and run the gel at 100V (expected initial current: 15–20 mA, expected final current: 5–10 mA) for 90min or stop the run when the dye front reaches the reference line imprinted on the bottoms of the cassettes. 7. Remove gel once run is complete ➢ After electrophoresis is complete, turn off the power supply and disconnect the electrical leads ➢ Remove the gels from the cell and pour out the running buffer ➢ Open cassette, by aligning the arrow on the opening lever with the arrows marked on the cassette – insert the lever between the plates and apply downward pressure to break each seal ➢ Gently remove gel 8. Stain gels for visualization using Coomassie Brilliant Blue R-250 Staining Solution #1610436 ➢ Once gel is removed, transfer into a clean staining tray ➢ Wash gel with 200mL of distilled water to remove SDS – gently shake for 5 min (discard water and repeat 3 times) ➢ Remove water add enough staining solution to cover the gel – allow to stain with gentle agitation for 1 hour ➢ Discard staining solution and add 200mL water – allow rinse with gentle agitation for at least 30min before visualization ➢ Protein bands are visualized as blue bands once the excess stain is removed 5 Expectations 1. Proteins were denatured and migrated through the polyacrylamide gel based on size ➢ Therefore, larger proteins will be present higher up in the gel and smaller proteins lower down in the gel 2. The proteins should appear on the gel as blue bands since staining with Coomassie was performed 3. There should be no band visible in the negative control and this confirms that any bands seen in the other lanes are actually that of the protein samples 4. Since one protein is loaded into each well, we expect 1 band to be visible in each well 5. The molecular weight marker will be visible containing 10 bands in total and we can create a standard curve based on distance travelled and protein size – we will then be able to estimate the size of the 5 proteins in this experiment by extrapolating from the standard curve 6 References Bio-rad Bulletin 6040 Rev A. (n.d.). Bio-rad.  Determining Molecular Weight of Unknown Protein Sampleshttps://www.biorad.com/webroot/web/pdf/lsr/literature/Bulletin_6040A.pdf Good, E. (2021). MCB 253, Experimental techniques in cellular biology. School of molecular and cellular biology. University of Illinois at Urbana-Champaign Nowakowski, A.B., Wobig, W.J., & Petering, D.H. (2014). Native SDS-PAGE: High Resolution Electrophoretic Separation of Proteins With Retention of Native Properties Including Bound Metal Ions. Metallomics, 6(5): 1068–1078. doi:10.1039/c4mt00033a Table 2- Moecular weight standard M.W. Lane Band 1 Band 2 Band 3 Band 4 Band 5 Band 6 M.W. (kDa) 250 200 150 100 50 37 Distance migrated (mm) 4.2 5.5 6.4 11 17.1 18.5 Table 3 – Sample Proteins Protein 1 lane Distance migrated (mm) M.W. (kDa) Band 1 Protein 2 lane Table 1 – Dilution Chart Initial concentration (ug/uL) Sample Volume (uL) lane 1 1.5 lane 2 1.5 lane 3 1.5 lane 4 1.5 lane 5 1.5 Buffer volume (uL) Note: 1. You are given 50 uL for each sample 2. Each protein sample has an initial concentration of 1.5 mg/mL 3. Each well holds 20 uL 4. Each gel has 10 wells. You will load 7 wells (2 MW + 5 Samples), and fill the rest of three wit 5. Coomassie blue can stain 1-5 ug of purified proteins, so aim your final amount of protein to b 6. Keep the final concentration column/final amount of protein same for all 5 lanes Create another table to indicate what will be loaded into each well Distance migrated (mm) M.W. (kDa) Total final volume (uL) Final Concentration (ug/uL) sample volume + buffer volume nd fill the rest of three with buffers nal amount of protein to be within this range. Final amount of protein (ug) final concentration x total final volume SDS-PAGE Data: Gel Image • • • • Use the gel image below to measure the distance migrated by each MW protein standard on the gel (from the bottom of the well (yellow line) to the center of each MW protein standard band. Determining Molecular Weight of Unknown Protein Samples

There are six protein bands total in the MW lane. In a data table, record the MW of each protein in the standard (in kDa, see MW standard information at the end of this document) and their corresponding distance migrated (in mm). Measure the distance migrated from the bottom of the well (yellow line) for each protein band in wells 1-5 (protein samples 1-5). Record your data in a table. Gel Image Key: Yellow line= bottom of the well MW= Molecular Weight Standard Lane 1 = Protein 1 Lane 2= Protein 2 Lane 3= Protein 3 Lane 4= Protein 4 Lane 5= Protein 5 1 MW Standard information: • There are six protein bands in the MW Standard lane in the gel image above. • The molecular weights for each protein in the molecular weight standard are listed to the right of each band in the image below (enlarged imaged of MW lane from the gel image above). • On semi-log paper (provided for you in the Appendix of your lab manual) or in Excel, plot the MW against the distance migrated for each protein standard band. This will create your standard curve. 250kDa 200kDa 150kDa 100kDa 50kDa 37kDa Analyzing the Data- Creating a Molecular Weight Graph (Distance migrated (mm) vs. Molecular weight (kDa)) To determine the molecular weight of each your proteins (1-5), do the following: 1. Measure the distance migrated by each MW protein standard on the gel (from the bottom of the well (yellow line) 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. Determining Molecular Weight of Unknown Protein Samples

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 protein sample have more than one band in a single well? 2 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. Determining Molecular Weight of Unknown Protein Samples

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.