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
– b-mercaptoethanol: strong reducing agent
• 2-ME, b-ME (or other reducing agents, eg. DTT)
P
P
reduction
S
+ 2H+ + 2e-
SH
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 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
Migration of band (cm)
Migration of dye front (cm)
Dye front
Rf =
Distance gel “ran”: dye front
Band
Relative migration/mobility (Rf)
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 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.
http://home.earthlink.net/~llica/wchmport.htm
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?