Lab discussion 1.Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) 2.Purification table 3.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 –  -mercaptoethanol: strong reducing agent 2-ME,  -ME (or other reducing agents, eg. DTT) P S S P +2H + + 2e - P SH P reduction

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 gel Soak 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 Distance gel “ran”: dye front Migration of band (cm) Migration of dye front (cm) Relative migration/mobility (Rf) R f = Band Dye front

Standard curve of R f values

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

Purification table

Formal report Let 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.

Formal report Let 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 report Let 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?