lab discussion

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

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 accordingto 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)

Rf =

Ban

d

Dye

fro

nt

Standard curve of Rf 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 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?