Protein purification and

Crystallisation
Data Collection
Exploit features of protein to purify
Solubilty - ‘salting out’
Charge - ion exchange chromatography
Size - gel filtration chromatography
Hydrophobicity – hydrophobic interaction Chromatography
Affinity Tags – affinity chromatography

SDS and native electrophoresis

Assay for specific activity, identity

Crystallisation

Prepare supersaturated solution of protein by adding

Precipitants, adjust pH, control temperature.

Enable nucleation – homogeneous or heterogeneous

Phase diagram of protein solubility showing ‘catch22’ – the level of supersaturation required for homogeneous
Nucleation higher than that required for sustained growth (metastable zone) – showers of little crystals.
Control with slow (hrs – months) approach to nucleation zone or seeding.

Protein purification and

crystallisation
How to find the right conditions – needle in a haystack problem?

Screen with arrays of conditions known to be effective.

Use robotic nanolitre dispensers (mosquito, honeybee etc).
Crystal hotels with photographic review.

MRC 96 well plate

 https://www.youtube.com/wh?v=MGnJ7X-ekug

Mosquito crystallisation robotic pipettor - delivers96 x 50nl drops in ~ 1 min

 Crystallisation of Membrane Proteins

Detergents for solubilising membrane proteins

Crystallisation of membrane proteins with detergents and lipidic cubic phase (LPC)
 LCP
Monolein + water + buffered protein + precipitant