Lets focus on proteinprotein interactions

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Lets focus on protein-proteininteractions
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Biochemical versus Genetic methods
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Do BEC-1 and CED-9 interact?
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Detection of protein-protein interactions by in
vitro pull-down assay
Purified glutathione S-transferase BEC-1 fusion
protein
Glutathion-coated beads
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BEC-1 interacts directly with CED-9
1. in vitro pull-down
2. co-immunoprecipitation
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Co-immunoprecipitation
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Protein A and G

• Many Gram-positive, pathogenic bacteria express
  cell surface proteins which are able to bind to
  immunoglobulins.
• Two of the first such proteins to be recognised
  were protein A, a cell wall component of
  Staphylococcus aureus and protein G, a protein
  associated with the cell wall from certain
  Streptococci.

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Detection of protein-protein interactions by
co-immunoprecipitation
CED-9 was detected by Western blotting in the
BEC-1/CED-9 complex
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Identification of LET-418, as an interacting
partner of MEP-1 Identification by Co- IP and
MALDI-TOF
The Nuclear Proteins MEP-1 and LET-418 Interact
In Vivo and Have Similar Developmental
Activities (A) MEP-1 and LET-418 interact in
vivo. Extracts prepared from wild-type (wt) and
from mep-1gfp transgenic animals were
immunoprecipitated with a GFP-specific monoclonal
antibody. Eluted proteins were subjected to
SDS-PAGE followed by silver staining. MEP-1GFP
protein migrating at 165 kDa and a second protein
(LET-418) migrating at 280 kDa were detected in
the transgenic strain, but not in wild-type
(wt) (as indicated).
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The principle of the yeast two-hybrid system
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A hybrid transcriptional activator can be
generated from the DNA-binding domain (DBD) of
the LexA bacterial transcription factor and from
the activation domain (AD) of the yeast GAL4
transcription factor
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Consruction of a bait fusion
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Construction of a prey fusion
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If bait and prey interact, DBD and AD restore the
functional TF, and it results in transcription of
reporter genes
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Construction of a bait plasmid
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Bait and prey (hunter) plasmids
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The principle of the membrane-based Y2H system
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Bait-construction in the membrane-based Y2H system
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Prey-construction in the membrane-based Y2H
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Interaction of bait and prey results in cleavage
and liberation of TF