Detection of Proteins

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Detection of Proteins

• SDS-PAGE, Immunoprecipitations, Western Blot
  Analysis, Immunofluorescence, Immunohistochemistry

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Polyacrylamide Gel Electrophoresis (PAGE)

• Theory
• Denatured proteins separate on the basis of size
• Polymers of acrylamide act as molecular sieve
• All proteins become negatively charged and move
  towards positive electrode

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PAGE (2)

• Procedure
• Preparation of polyacrylamide gel
• Preparation of sample
• Electrophoresis
• Detection of proteins bands

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PAGE (3)

• Advantages
• High resolution due to sharp bands
• High sensitivity
• Reproducibility
• Speed (1-2 hrs to prepare 3-6 hrs to run 2 hrs
  to stain 6-12 hrs to destain

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Page (4)

• Uses
• Determination of molecular weight
• Purification of proteins
• Sequencing
• Recovery from gels
• Structural analysis
• Immunoprecipitations
• Western blots

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PAGE (5)

• Artifacts
• Aggregation due to insufficient denaturation of
  disulfide bridges
• Degradation by proteases
• Curved bands due to protein-glass interactions,
  uneven gel surface or overheating
• Disadvantages
• Polyacrylamide is a neurotoxin

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PAGE (6)

• Types of Gels
• SDS-PAGE linear and gradient
• Isoelectric focusing
• 2-D Gel electrophoresis

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Isoelectric focusing
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2-D Gel electrophoresis
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Immunoprecipitations

• Antigen-antibody reaction
• Can be very specific depends on antibody
  specificity
• Can be used to identify and purify protein
• Can be used to detect protein-protein
  interactions (Co-Immppt).
• Immune ppt protein can be detected by western
  blot or autoradiography

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Western Blot Analysis

• Separate proteins by SDS-PAGE
• Transfer protein electrophoretically to membrane
• Incubate membrane with specific antibody
• Detect protein-antibody interaction by
  radioactive or non-radioactive methods
• Can be used to detect protein or to detect
  presence of antibody

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Immunofluorescence Assay (IFA)

• Detect specific proteins expressed in tissues or
  cells
• Detect specific antibodies (e.g. anti-viral)
• Very sensitive requires use of fluorchrome
• Can be time consuming
• Results can be subjective (eye of the beholder)
• Results are short lived
• Requires several negative controls

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Induced BCBL-2 cells expressing vIL-6
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BCBL-1 cells expressing LANA-1
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Immunohistochemistry

• Same principle and use as IFA
• Uses enzyme-linked antibodies
• Less sensitive than IFA, but long term results
  are possible

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Figure 1 HHV-8 protein expression A. Staining
for LANA-1 40x, 100x inlay B. Staining for
LANA-1 40x, 100x inlay
C. Staining for K8.1 40x, 100x inlay
D. Staining for vIL-6 40x, 100x inlay