Proteins separation and analysis using Fast Protein Liquid Chromatography Ayelet David, Ph.D Dept. of Clinical Pharmacology

Fast Protein Liquid Chromatography (FPLC) Column chromatography used to separate or purify proteins from complex mixtures based on size, charge distribution, hydrophobicity of biorecognition (affinity chromatography). Typical columns used for protein purification, include: Gel Filtration Chromatography — separate proteins according to their size. Also termed as “size exclusion chromatography” (SEC). Ion exchange chromatography — separate proteins based on surface-charges. Reversed phase or hydrophobic interaction — separates based on hydrophobicity. Affinity chromatography — separates based on ligand affinity, such as a His-tagged protein would use a nickel column.

Gel Filtration Chromatography - Gel Filtration Chromatography - Principle Molecules elute in order of size. The largest molecules come first ; other molecules leave the column in decreasing order of size ; the smallest ones come last.

Stationary phase pore Crossed-linked matrix Stationary phase: porous, cross-linked beads (dextran, agarose, polyacrylamide) degree of cross-linking determines diameter of pores and fractionation range of biomolecules of different size proteins do not attach to column

Scanning electron micrograph of an agarose gel, Magnification x 50,000 The chromatographic medium is a gel. The gel is in the form of beads. Ref: Anders S. Medin, PhD Thesis, Uppsala University 1995

Biomolecules are separated according to size (hydrodynamic volume)

A gel beads that encloses an internal solvent space. Smaller molecules (red) can freely enter the internal solvent space of the gel bead, whereas larger molecules (blue) are too large to penetrate the gel pores. Separation according to size

9 Void volume V o Volume of the gel matrix V g Pore volume V i V o = Void volume V e = Elution volume within the separation range of the gel V i = Inner pore volume = V c - V g - V o V c = Total (geometric) volume of the column V t = Total volume of the column VoVo VeVe VtVt VcVc Terms and explanations

10 The void volume V o VoVo VeVe Volume Concentration Elution volume for very large molecules, V o All molecules larger than the largest pores will elute together at the void volume. V e = V o For most gel filtration columns, the void volume represents 30 to 40 % of the total column volume V c.

11 V t and V c Volume Concentration VtVt VcVc VoVo VeVe Elution volume for very small molecules, V t Geometric volume of the gel bed, V c The volume in which a small molecule elutes from the column is V t V t = V o + V i V t is slightly smaller than V c.

12 The distribution coefficient, K d K d is difficult to get because V i is difficult to measure K d = the fraction of the stationary phase which is available to a given solute. It depends only on the gel and the size of the solute.

13 The coefficient K av K av is easy to get and it is more useful in practice K av is not a true partition coefficient Since the V i is difficult to measure, it is usual to substitute the term (V t -V o ) for V i in the partition equation and call the result K av instead of K d.

14 K av for very large and very small molecules

15 K av should always be in the range 0 to 1 VtVt VeVe Volume Concentration Elution volume when K av = 1 Elution with K av > 1 Adsorption has occurred

Some typical applications for gel filtration 1.Group separation: Desalting, Buffer exchange, Removing reagents 2.Purification of proteins and peptides: complex samples, monomer/dimer 3.Estimation size & size homogeneity

Sephadex G-25 is widely used for these applications. Its separation range makes it suitable for group separations work, such as the removal of salt contaminants from molecules larger than about 5,000. Application 1 - Application 1 - Desalting and buffer exchange (Sephadex G-25)

Application 2 - Application 2 - Purifications of proteins A gel filtration column with two different size molecules applied. The larger molecules exit the column first

Separating dimer and oligomers from monomer

There is a sigmoidal relationship between Kav and the logarithms of the molecular masses for molecules of similar shape. Over a considerable range, a linear relationship exists. Application 3 - Application 3 - Determination of molecular weight

A selectivity curve is fairly linear between K av values of 0.1 and 0.7 The molecular weight range which lies between these values is defined as the useful fractionation range of the medium.

The exclusion limit is the molecular weight of the smallest molecule which cannot enter the pores of the matrix. It is an extrapolated value defined by convention.

Gel matrix has optimum ~ linear range

AKTA FPLC System All instrument settings and functions are under the direct control of UNICORN, a real-time control system. Syringe pump producing accurate, reproducible, pulse free flow rate and a precise gradient formation On line monitor offering the possibility to measure UV conductivity, and pH. Fraction collector A seven port motorized valve, used for sample application Injection valve INV-907 A single chamber mixer, powered and controlled from Pump P-920 Mixer M-925 Column Column is easily placed on the outside of the system UV detector

All data from a run, including running conditions, method and start protocol, and a complete log of every event during the run, are automatically stored in a single file.

Efficiency is governed by the separation medium, how well the column has been packed, and the running conditions. Selectivity is governed almost entirely by the separation medium itself, so choosing the right medium is essential to obtaining the right selectivity for a given separation