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SDS-PAGE, protein analysis method (Sodium Dodecyl Sulfate-Polacrylamide Gel Electrophoresis)

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1983 Pulsed field gel electrophoresis. 1983 Capillary electrophoresis introduced. TriColor Pre-stained Protein Molecular Weight Markers * Title: PowerPoint – PowerPoint PPT presentation

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Title: SDS-PAGE, protein analysis method (Sodium Dodecyl Sulfate-Polacrylamide Gel Electrophoresis)


1
SDS-PAGE, protein analysis method (Sodium
Dodecyl Sulfate-Polacrylamide Gel
Electrophoresis)
Dec 6th, 2011
2
????
  • 1. ????????????
  • SDS-PAGE????
  • 3. ???SDS-PAGE?????????
  • 4. Western Blot (??????)????????
  • 1.

3
  • Protein characterization (homogeneity)
  • Protein concentration determination.
  • SDS-PAGE (size)
  • IEF(pI)
  • 2 dimensional gel
  • Western blot analysis
  • Limited N-terminal amino acid sequence
  • (Edman degradation)
  • 7. Catalytic activity (kinetic parameters Km,
    Vmax,
  • Ka or Ki)
  • 8. Protein crystallization
  • 9. Peptide mapping

4
How much protein?
Amino acid analysis
Spectrophotomeric colorimetric
electrophoresis methods
methods
(1-D or 2-D)
A280 A205 fluorescence Bradford
Lowry emission methods
methods
India ink Iron stain Gold stain Antibodies (wes
tern blotting)
Coomassie blue stain Silvern stain
detection
electroblotting
detection
5
Is the protein pure? How many protein Subunits?
What is Mr of protein and its subunits?
chromatography
Electrophoresis (1-D and 2-D)
detection
conventional
High performance Liquid chromatography
Size-exclusion
Silver stain
Comassie Blue stain
Gel filtration
Comparision with molecular sizes for standard
proteins
Estimate of Mr of unknown protein
Estimation of Mr of unknown protein And its
subunits determination of number Of protein
subunits
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  • Electrophoresis
  • Brief history
  • 1930s first report of the use of sucrose for gel
    electrophoresis.
  • 1955 Introduction of starch gels, not very good
    separation.
  • 1959 Introduction of acrylamide gels for first
    time (Raymond and Winstraub), accuracy of control
    of parameters such as pore size and stability.
  • Disc gel electrophoresis by Ornstein and Davis.

14
  • 1969 Introduction of denaturing agents especially
  • SDS separation of protein subunit (Beber and
    Osborn).
  • 1975 2-Dimensional gels Ofarrel isoelectric
    focussing
  • then SDS gel electrophoresis.
  • 1977 Sequencing gels.
  • Late 1970s agarose gels.
  • 1983 Pulsed field gel electrophoresis.
  • 1983 Capillary electrophoresis introduced.

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Recognition of basic components that used in
SDS-PAGE
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Applications of SDS-PAGE
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TriColor Pre-stained Protein Molecular Weight
Markers
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No signal in Western blotting
Poor antibody or insufficient antibody
Insufficient protein loaded onto the gel
Protein did not transfer Reagents
improperly prepared Specificity of
secondary antibody not compatiable Presence
of azaide or other preservatives in buffer
Sour protein samples
No
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  • Excess signals in Western blotting
  • Insufficient washing or blocking
  • Insufficient dilution of antibody
  • Excessive antibody incubation time
  • Excessive protein loaded on to gel
  • Excessive reaction development times
  • Post-translational modification
  • Protein isoforms (multiple genes or alternative
    splicing)
  • Sour protein samples
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