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2 Ion Exchange Chromatography

3 Ion chromatography is used for the separation and analysis of ions (both anions and cations). The mode of separation is called ion exchange and it is based on the premise that different sample ions migrate through the separator column at different rates, depending upon their interactions with the ion exchange sites of the packing material (ion-exchange resins bonded to inert polymeric particles). The ion-resin interaction is unique and characteristic for each ion for a given resin. By comparing the data obtained from a sample to those obtained using known standard solutions, sample ions can be identified and quantitated. The common detector is a conductivity cell, which measures the electrical conductance of the sample ions. Amperometry, UV-VIS absorbance, photodiode array, and mass spectrometry detectors can also be used. Affinity Chromatography Filtration Gel Chromatography

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6 Purification Make your protein as pure as possible One single band on an overloaded gel Make sure that no aggregates are present, or mixtures of e.g. monomers and dimers (check with e.g. dynamic light scattering) Concentrate your protein to about 10 mg/ml Do not use high buffer and salt concentrations in your final prep for crystallization Researchers have been successful with crystallizing His-tagged proteins (Dijkstra 2006)

Crystallization Hanging drop method Sitting drop technique Batch crystallization under oil Drop contains 1:1 (or 2:1) protein:precipitant Crystallization screens Crystal Screens 1 and 2 Wizard screens 1 and 2 PEG screens Additive screen Beware: calcium + phosphate easily gives calcium-phosphate crystals! 7

8 What is a good crystal? Dimension 0.2 x 0.2 x 0.2 mm No scratches or weird outgrows Sharp edges It should diffract to high resolution It should give a “clean” diffraction pattern It should have a low “mosaicity” Aquifex aeolicus amylomaltase Best condition: 100 mM Tris 7.5, 200 mM A.S., 35% MPD (condition 11)

9 Crystals Are built up in 3 dimensions of repeating units (unit cell) in each unit cell exactly the same atoms are present at exactly the same positions Crystals behave like a grating the atoms in a crystal can be considered to be the grooves in a three-dimensional grating The distances between the atoms is about 0.15 nm (0.15 x m) With normal visible light (  = 400 – 650 nm) such details are not visible, but with X-rays (  = 0.1 nm) they are!

10 How about bio-macromolecules? They can also be crystallized They have a size of only mm They contain about 50% water They diffract weakly -> strong X-ray sources needed!

11 X-ray diffraction of protein crystals Xray diffraction patterns have symmetry This symmetry is caused by the symmetry in the crystal There are exactly 230 ways how to combine symmetry operations (2,3,4,6-fold rotations, translations, inversions, mirrors) (space groups) For proteins only 75 possibilities are allowed (No mirrors or inversions: L-amino acids -> D-amino not allowed

12 With structure factors and phases…. We can calculate the distribution of the electrons in the unit cell (= the electron density distribution)! Model building

13 Result of X-ray diffraction Why is protein crystallography useful? X-ray diffraction experiments yield – a better understanding of the biochemistry of processes in living organisms – a better understanding of the molecular basis of some diseases – new medicines for some diseases – new enzymes for industrial applications –...