METHODS FOR SHOWING INTERACTIONS

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• METHODS FOR SHOWING INTERACTIONS
• 1. Between protein and DNA
• EMSA (electrophoretic mobility shift assay), gel
  shift

• 2. Between protein and another protein
• immunoprecipitation (in vivo)
• GST pull-down assay (in vitro)
• yeast two-hybrid system (in yeasto)

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EMSA
DNA protein
control oligos
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EMSA
DNA protein
antibody
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Jun, Fos (AP-1) and MBF1 are needed to form a
complex with DNA
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• METHODS FOR SHOWING INTERACTIONS
• 1. Between protein and DNA
• EMSA (electrophoretic mobility shift assay), gel
  shift

• 2. Between protein and another protein
• immunoprecipitation (in vivo)
• GST pull-down assay (in vitro)
• yeast two-hybrid system (in yeasto)

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Immunoprecipitation

• affinity purification based on isolation of Ag-Ab
  complexes
• analyze by gel electrophoresis
• initially based on centrifugation of large
  supramolecular complexes
• high and ?equal amounts
• isolation of Ag-Ab complexes
• fixed S. aureus
• protein A-agarose
• protein G-agarose

• Bacterial proteins that bind IgG (Fc)
• protein A (Staphylococcus aureus)
• protein G (Streptococcus)
• binds more species and subclasses

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Typical IP Protocol

• 1. Solubilize antigen
• usually non-denaturing
• SDS excess of TX100
• 2. Mix extract and Ab
• 3. Add protein G-agarose, etc
• 4. Extensively wash
• 5. Elute with sample buffer
• 6. SDS-PAGE
• 7. Detection
• protein stain
• radioactivity

G
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Tracheae defective/Apontic is an MBF1 partner
Immunoprecipitation
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• increase stability
• affinity purification
• detection/assay
• spectrophotometric
• binding assays
• antibodies
• export signals

Fusion Proteins
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1. Engineered protease site allows removal of
fusion partner
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2. Addition of a few residues should have minimal
effect on recombinant protein

• His6 Tag
• add 6 consecutive His to either end
• binds metals

• Epitope Tag
• 6-12 amino acids
• mAb for detection or purification

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Fraction number
35
25
pure protein
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Sepharose GSH
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Sepharose GSH
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Tracheae defective/Apontic is an MBF1 partner
GST pull-down
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• METHODS FOR SHOWING INTERACTIONS
• 1. Between protein and DNA
• EMSA (electrophoretic mobility shift assay), gel
  shift

• 2. Between protein and another protein
• immunoprecipitation (in vivo)
• GST pull-down assay (in vitro)
• yeast two-hybrid system (in yeasto)

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Yeast 2-Hybrid Assay
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Yeast two-hybrid assay
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METHODS FOR SHOWING INTERACTIONS IN
CELLS Between transcription factors and target
genes Between transcription factors and their
coactivators/repressors Between nuclear
receptors and their ligands
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How to measure gene activation in eukaryotic
cells ?
Transfection assays
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How to measure gene activation in eukaryotic
cells ?
Transfection assays