Understanding SDS-PAGE: Principles, Techniques, and Applications in Protein Analysis
This discussion provides an overview of denaturing polyacrylamide gel electrophoresis (SDS-PAGE), a critical technique used in biochemistry for separating proteins based on size. It outlines the preparation of protein samples, including denaturation and the role of SDS and reducing agents. Key steps such as loading samples, running the gel, and staining for visualization are described. Additionally, the importance of estimating protein size using standard curves and the implications for protein purity assessment are discussed, ensuring accurate interpretation of SDS-PAGE results.
Understanding SDS-PAGE: Principles, Techniques, and Applications in Protein Analysis
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Presentation Transcript
Lab discussion • Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) • Purification table • Paper
SDS-PAGE • Separate proteins according to size • Here: actual size, not effective size as for gel filtration/size exclusion • Goal: visualization (typically not a purification step) • See protein’s purity • Calculate protein’s size
Preparation of protein sample • Denature all of the proteins (lose 2°, 3°, 4°) • Add strong detergent (SDS) • Heat • Break weak bonds: esp. hydrophobic interactions • b-mercaptoethanol: strong reducing agent • 2-ME, b-ME (or other reducing agents, eg. DTT) P P reduction S SH 2e- + 2H+ + S SH P P
Preparation of protein sample • 1° structure: protein’s charge depends on pH • Different proteins migrate differently in electrical field • Additional role of SDS: ‘coat’ proteins uniform negative charge
Preparation of protein sample • Components of sample buffer • SDS • Buffer: constant pH • Glycerol: add density: samples ‘sink’ in the wells • Blue dye: doesn’t bind proteins (proteins remain invisible for now) • Allows tracking of gel’s progress
“Running” the gel Proteins migrate through ‘pores’ in a polymer according to an electrical gradient The smaller the protein, the easier it can ‘snake’ through the pores
“Stain” the gelSoak the gel in a dye that selectively binds protein (Coomassie) Larger proteins Smaller proteins
Final product “Standards”/”Markers” Allow estimation of unknown protein’s size
Size estimation: standard curve Relative migration/mobility (Rf) Migration of band (cm) Rf = Migration of dye front (cm) Band Dye front Distance gel “ran”: dye front
SDS-PAGE gives an estimate of protein size • Highly charged proteins • Proteins retaining some 2° and 3° or even 4° structure • Measuring of mobilities is an inexact science • Try to measure to the ‘fattest’ part of the band
Formal reportLet me be your (possibly wrong) grammar teacher for the day • “That” vs. “Which” • That: restrictive. The ‘that’ phrase is necessary for the sentence to make sense. • Little activity was retained by the fumarase that was stored at -20°C. • Little activity was retained by the fumarase which was stored at -20°C. • Which: descriptive. The ‘which’ phrase adds to the sentence but could be omitted. • We determined the pH dependence of fumarase, which works via an acid-base mechanism. • We determined the pH dependence of fumarase. • The pH dependence of fumarase was determined, which works via an acid-base mechanism. http://home.earthlink.net/~llica/wchmport.htm
Formal reportLet me be your (possibly wrong) grammar teacher for the day • “This” and “These” • always need an object • The spectrophotometer began to release a substantial amount of black smoke. This suggests that huge mistakes were made. • Blah, blah, blah… These data suggest that my partner is brain dead.
Formal reportLet me be your (possibly wrong) grammar teacher for the day • No (few) apostrophe’s! • Don’t use contractions • are not vs. aren’t • Try to avoid words’ possessive forms. • “The color of the solution…” instead of “The solution’s color…” • Don’t use an apostrophe to make word’s plural. • “pH values” instead of “pH’s” or “pHes”
Report • Abstract • Motivation, question, brief strategy, brief results, conclusion • Concise! • Intro • Why are you asking this question? • How does previous research inform this work? • Motivation, question, strategy… leave out results, conclusion • Discussion • What does this work mean? • How does this work inform future research?
