This is the path to enlightenment for VDS researchers. It may be a direct path or a random walk for you!

These are in general order - but steps can be done in parallel

MILESTONES

Clone your Coding DNA Sequence (CDS) into expression vector
  • Primary PCR
  • Secondary PCR
  • PCR^2
  • PCR cleanup, Nanodrop
  • Cloning protocol
    • Cut pNIC-Bsa4 (can be done once you have PCR^2 working)
    • Annealing & Transformation
    • Grow up clones
    • DNA sequencing for positives
    • Grow up positives and Midiprep, then archive

Virtual Screening of your target (should be done in parallel with wet lab - i.e. not sequentially)
  • Molprobity on structure
  • Create Homology Model if needed (run Molprobity on this)
  • Pick Control Ligands
  • LigPrep Control Ligands
  • Set up protein in GOLD & Dock control ligands
  • Analyze control docking
  • Dock screening libraries of novel compounds
  • Order top hits (once you have soluble protein from wet lab)

Express Protein
  • Transform plasmid into expression cells
  • Express protein
  • Harvest & Purify protein through Nickel affinity column and Size Exclusion (FPLC)
  • Characterize protein on gel
  • Store protein (2 ways)

Enzyme Assays
  • Test out if enzyme is active (Vary enzyme)
  • Vary substrate assays (determine Km value)

Inhibition Assays
  • Test out different concentrations of compounds ordered
  • Determine IC50 if it is a good inhibitor





Extra Toppings:
Virtual screening with ICM docking software
Ligand based virtual screening
Fluorescence melt assays
Crystallization trials

Extensions:
carry out MM-PB(GB)SA calculations of Free Energy of Binding to re score top ligands
- use Prime in Maestro Suite from Schrodinger