Proteins separation and analysis using Fast Protein Liquid Chromatography

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

2. 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.

3. 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.

4. pore Crossed-linked matrix Stationary phase 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

5. Ref: Anders S. Medin, PhD Thesis, Uppsala University 1995 • • 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.

7. Biomolecules are separated according to size (hydrodynamic volume)

8. Separation according to size 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.

9. Vo= Void volume Ve = Elution volume within the separation range of the gel Vi = Inner pore volume = Vc- Vg -Vo Vc = Total (geometric) volume of the column Vt = Total volume of the column Void volume Vo Ve Volume of the gel matrix Vg Vo Vt Pore volume Vi Vc 3 1 2 Terms and explanations

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

11. Vt and Vc Elution volume for very small molecules, Vt Concentration Geometric volume of the gel bed, Vc Vo Volume Ve Vt Vc The volume in which a small molecule elutes from the column is Vt Vt = Vo + Vi Vt is slightly smaller than Vc.

12. The distribution coefficient, Kd Kd= 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. Kd is difficult to get because Vi is difficult to measure

13. The coefficient Kav Since the Vi is difficult to measure, it is usual to substitute the term (Vt-Vo) for Vi in the partition equation and call the result Kavinstead of Kd. Kav is easy to get and it is more useful in practice Kav is not a true partition coefficient

14. Kav for very large and very small molecules

15. Kavshould always be in the range 0 to 1 Elution with Kav > 1 Adsorption has occurred Concentration Elution volume when Kav = 1 Volume Ve Vt

16. Some typical applications for gel filtration • Group separation: Desalting, Buffer exchange, Removing reagents • Purification of proteinsand peptides: complex samples, monomer/dimer • Estimation size & size homogeneity

17. (Sephadex G-25) Application 1 - Desalting and buffer exchange 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.

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

19. Separating dimer and oligomers from monomer

20. Application 3 - Determination of molecular weight 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.

21. A selectivity curve is fairly linear between Kav 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.

22. 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.

23. Gel matrix has optimum ~ linear range

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

25. 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.

26. 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