lab discussion
DESCRIPTION
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) - PowerPoint PPT PresentationTRANSCRIPT
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?