Protein purification techniques

Protein purification techniques

1. Protein Purification Techniques Navaira Arif Roll No. 18

2. The basic aim in protein purification is to isolate one particular protein of interest from other contaminating proteins to study its structure and function, increasing its stability and large scale production

3. Protein Purification Purification of proteins i s an essential first step in understanding their f unction Proteins must be released from the cell to b e p urified Based on the basic properties of proteins like solubility , size , charge and binding affinity

4. General Steps in Protein Purification Selection of a protein source Assay of Proteins Homogenization and Solubilization Stabilization of Proteins Detergent e.g. Triton X 100 pH 7 and temperature below 25°C Radioimmunoassay, ELISA, Western Blotting

5. 1. Ammonium Sulfate Precipitation This technique exploits the fact that the solubility of most proteins is lowered at high salt concentrations The concentration of ammonium sulfate at which a particular protein comes out of solution and precipitates is different from other proteins in the mixture

6. 2. Dialysis A semipermeable membrane is used to remove small molecules such as salts and ammonium sulfate from a protein solution Based upon size of molecules

7. 3. Gel Filtration Chromatography separate proteins according to their size. Also termed as “size exclusion chromatography” A gel filtration column has beads with channels running through them e.g. agarose A good gel for gel filtration contains about 95% water

8. Smaller molecules can freely enter the internal solvent space of the gel bead Larger molecules are too large to penetrate the gel pores and travel between beads and elute first

9. 4. Ion exchange chromatography separate proteins on basis of their over all (net) charge Retention is based on electrostatic interaction between solute ions and fixed ion charge on the stationary phase

10. Anion exchange resins (positive charge) separate negatively charged compounds Cation exchange resins (negative charge) separate positively charged compounds

11. 5. Affinity chromatography Based upon molecular conformation E xploits the specific, high affinity, non-covalent binding of a protein to another molecule, the ligand Ligands function in a fashion similar to that of antigen-antibody interactions

12. The ligand is immobilized onto a solid support matrix The crude extract is passed through the column. The target molecule for which the ligand possesses affinity is retained All other material is eluted The bound target protein is eluted by alteration of the mobile-phase conditions.

13. 6. Fast Performance Liquid Chromatography A type of liquid chromatography where the flow rate of the solvent is set through computer input and controlled by pumps The chromatographic bed is composed by the gel beads alone when they are inside the column The sample is introduced into the injector and then carried into the column by the flowing solvent

14. Once in the column, the sample mixture separates as a result of different components adhering to or diffusing into the gel in form of different zones called “Bands”

15. 7. Gel Electrophoresis When placed in an electric field, proteins (having net charge) will move towards one electrode or the other The greater the net charge the faster the molecule will move PAGE – Polyacrylamide Gel Electrophores is ( carried out in a gel which serves as a molecular sieve)

16. Protein samples are loaded into sample wells An electric field is applied across the gel from top to bottom Proteins migrate down through the gel The smaller the protein the further it will migrate

17. SDS- PAGE P rotein mixture is heated in the presence of 2-mercaptoethanol and SDS U nfolded polypeptide chains will then migrate towards the anode Smaller polypeptides migrate further through the gel than larger ones

18. Proteins are purified from a mixture using combination of several techniques based on protein properties