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Lab discussion

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Lab discussion Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) Purification table Paper – PowerPoint PPT presentation

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Title: Lab discussion


1
Lab discussion
  1. Denaturing polyacrylamide gel electrophoresis
    (SDS-PAGE)
  2. Purification table
  3. 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 proteins purity
  • Calculate proteins 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 proteins 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 doesnt bind proteins (proteins remain
    invisible for now)
  • Allows tracking of gels 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 proteins 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 -20C.
  • Little activity was retained by the fumarase
    which was stored at -20C.
  • 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) apostrophes!
  • Dont use contractions
  • are not vs. arent
  • Try to avoid words possessive forms.
  • The color of the solution instead of The
    solutions color
  • Dont use an apostrophe to make words plural.
  • pH values instead of pHs 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?
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