Buffer Preparation: Protein Purification

Topic 2 1. Preparation for Protein Purification: Buffer solution & Additives  What is a buffer solution? A buffer solution (more precisely, pH buffer or hydrogen ion buffer) is an aqueous solution consisting of a mixture of a weak acid and its conjugate base, or vice versa. I...

Topic 2 1. Preparation for Protein Purification: Buffer solution & Additives  What is a buffer solution? A buffer solution (more precisely, pH buffer or hydrogen ion buffer) is an aqueous solution consisting of a mixture of a weak acid and its conjugate base, or vice versa. Its pH changes very little when a small or moderate amount of strong acid or base is added to it and thus it is used to prevent changes in the pH of a solution. Buffer solutions are used as a means of keeping pH at a nearly constant value in a wide variety of chemical applications.  Requirements of Biological buffer 1. Solubility: Freely soluble in water but not other solvents. Allows ease in preparing concentrated stock of (10X, 50X, 100X etc.).pH of sodium phosphate may increase with dilution, 6.7 to 6.9 with 10-fold dilution and to 7.0 with 100- fold dilution 2. Ionic strength: Shouldnt alter protein structure or activity. Protonisation and deprotonisation highly dependent on ionic strength. Ionisation of amino acids can affect the structure and activity of a protein 3. Complex formation: When buffer complexes with metal ions, it causes protons to be released therefore reducing pH forms insoluble precipitates which can complicate purification. Phosphates precipitate with bivalent metal ions 4. Inert substance: Does not react with proteins which can affect activity of the protein 5. UV absorbance: Should not absorb any light longer than 230 nm as many protein based spectrophotometric investigations are performed at this range 6. Purity: Contamination can affect protein stability and activity 7. Costs: Purification requires large amounts of buffer therefore the buffer used should be cost-effective Preventing contamination in buffer preparation: Microbial contamination: Chemical impurities: Use high grade water, double distilled or millipore water Additives: Extending protein stability Depending on the target protein, it may be necessary to add compounds to the lysis buffer: to improve the stability of the target protein. to keep the protein in solution. E.g: -Protease inhibitors, EDTA (Ethylenediamine tetraacetic acid), PMSF (Phenylmethylsulfonyl fluoride) - Metal chelators - Reducing agents - Glycerol Buffer Preparation Video