The Protein Interactions Expert



Loss of Affinity Mutants

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A Loss of Affinity Mutants (LAM) screening yields interaction-deficient point mutants of your bait protein for a given partner. LAM takes advantage of our ULTImate Y2H™ technique.

Use LAM screening to map interaction hotspots and obtain dominant negatives to speed up your functional studies!

CONTACT US and submit the sequences of your proteins.

Applications

  • Identify critical amino acids required for your favorite interaction
  • Correlate loss of interaction with loss of function in cells

Molecules tested

  • Full-length proteins or fragments, peptides
  • Cytoplasmic or extracellular proteins
  • Loops and tails of membrane proteins

How does it work?

Mutants are generated by PCR and the experimental conditions are adapted to each target gene to get on average a single mutation per clone.

The mutant library is then screened against the interacting partner by yeast two-hybrid using the LacZ reporter gene. White or light blue yeast clones in which no or a weak interaction occurs are selected.

192 clones are fully sequenced to identify their mutation. Their phenotypes are confirmed in a secondary, semi-quantitative LacZ assay.

You select up to 10 point mutants which are further characterized in a quantitative colorimetric interaction assay. You then receive DNA plasmids for these 10 mutants for your own susbsequent functional studies.

Key benefits

  • Powerful point mutant screening method based on ULTImate Y2H™ yeast two-hybrid
  • Deeper understanding of your interaction and the amino acids involved
  • Generation of invaluable tools for functional studies in cells or in vitro
  • Rapidly conducted further to an ULTImate Y2H™ screen or a 1-by-1 Y2H assay

Deliverables

  • Pictures of the solid LacZ assay
  • Nucleic and proteic sequences of 192 mutant clones
  • Quantitative colorimetric LacZ results for 10 selected mutants, ready for publication
  • Delivery of DNA plasmids of up to 10 mutants
  • Detailed experimental methods
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