Lab discussion

1. Lab discussion • Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) • Purification table • Paper

2. 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

3. 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

4. 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

5. 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

6. “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

7. “Stain” the gelSoak the gel in a dye that selectively binds protein (Coomassie) Larger proteins Smaller proteins

8. Final product “Standards”/”Markers” Allow estimation of unknown protein’s size

9. 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

10. Standard curve of Rf values

11. 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

12. Purification table

13. 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

14. 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.

15. 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”

16. 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?