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Protein Analysis - PowerPoint PPT Presentation


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Protein Analysis. Techniques. SDS-PAGE Western Blotting MALDI SELDI ELISA X-ray crystallography NMR. SDS-PAGE. Sodium Dodecyl Sulfate – Polyacrylamide Gel Electrophoresis SDS Detergent Denatures proteins; native conformations disappear and only primary structures are retained

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Presentation Transcript
techniques
Techniques
  • SDS-PAGE
  • Western Blotting
  • MALDI
  • SELDI
  • ELISA
  • X-ray crystallography
  • NMR
sds page
SDS-PAGE
  • Sodium Dodecyl Sulfate – Polyacrylamide Gel Electrophoresis
  • SDS
    • Detergent
    • Denatures proteins; native conformations disappear and only primary structures are retained
  • PAGE
    • Uses polyacrylamide instead of agarose
    • Separation of protein fragments according to size
western blotting
Western Blotting
  • Similar to northern and southern blotting but analyzes proteins instead of nucleic acids
  • Uses modified antibodies as probes
    • Enzyme-linked
maldi and seldi
MALDI and SELDI
  • Mass spectrometry
  • Matrix–Assisted Laser Desorption–Ionization
    • Proteins are immobilized in a matrix
  • Surface–Enhanced Laser Desorption–Ionization
    • Modification of the MALDI
    • Simpler steps for preparation
elisa
ELISA
  • Enzyme-Linked Immunosorbent Assay
  • Qualitative or quantitative protein analysis
  • Two types of antibodies are used
    • Primary binds to specific antigen
    • Secondary binds to antigen-primary antibody complex
      • Enzyme-linked; produces colorimetric reaction in the presence of substrate
x ray crystallography
X-ray crystallography
  • The ultimate technique in determining protein structure
  • A protein crystal is created then bombarded with an X-ray beam to create a diffraction pattern
  • The electron cloud and correspondingly, the structure, of the protein is calculated from the generated diffraction pattern
nuclear magnetic resonance
Nuclear Magnetic Resonance
  • NMR utilizes differences in the spin states of the nuclei of atoms found in the sample
  • Protein NMR is very complicated and can be used to elucidate the atomic backbone of the protein.
  • Utilizes computer algorithms to generate a partial 3-D structure of the protein