Protein Purification and Analysis Solubility of proteins important for purification: 60-80% soluble, 20-40% membrane Size of proteins varies Some proteins expressed at high levels (collagen, hemoglobin) Some proteins expressed at low levels (repressors, signaling) Steps of purification and analysis (1) Choose protein to purify (2) Choose source (natural or expressed) (3) Soluble in aqueous solution?? (problem with membrane proteins) (4) Stability (5) Purify - based on some characteristic of protein (6) Study (activity, structure, mechanism of action, etc.)

Protein Purification and Analysis Characteristic:Procedure: Charge1. Ion exchange chromatography 2. Electrophoresis 3. Isoelectric focusing Size:1. Dialysis and ultracentrifugation 2. Gel electrophoresis 3. Gel filtration (size exclusion) chromatography 4. Ultracentrifugation Specificity:1. Affinity chromatography Polarity:1. Adsorption chromatography 2. Paper chromatography 3. Reverse-phase chromatography 4. Hydrophobic chromatography

Protein Purification and Analysis Chromatography - widely used to separate proteins, important purification technique for last 40 years BIG field of biochemistry deals with purification of proteins to study structure and function Column chromatography used to isolate proteins Mix of proteins loaded onto column that contains a matrix/resin Separation occurs because proteins interact with matrices/resins in different ways

Protein Purification and Analysis Chromatography Important steps in chromatography 1. Pack column - Column is packed with material (resin) that can absorb molecules based on some property (charge, size, binding affinity, polarity) 2. Equilibrate column - Column is washed with several column volumes of buffer 3. Load sample - apply sample mix to column 4. Wash column - Molecules washed through the column with buffer 5. Collect fractions - Fractions are taken, at some point your molecule will elute

Protein Purification and Analysis Size exclusion (gel filtration) chromatography Separate by size Column packed with porous beads As wash with buffer: Small molecules enter the beads Large molecules move between the beads Elution: Large proteins elute first, small proteins last

shape of a protein, its quaternary structure and other associated proteins will affect its apparent size in solution not recommended for separating proteins with only a small difference in molecular weight. choice of a chromatography medium is an important consideration in gel filtration Column matrixExclusion/fractionation range Sephadex G kD Sephadex G kD Sephadex G kD Sephadex G kD Sephadex G kD Bio-Gel P kD Bio-Gel P kD Bio-Gel P kD Bio-Gel P kD kD - kilodaltonD - dalton, g/mol matrices are gels of polysaccharides (dextrans) that are formulated into beads, each different matrix have different beads with varying degrees of crosslinking of the dextrans, swell beads to form pores different crosslinking = different pore sizes Protein Purification and Analysis Size exclusion (gel filtration) chromatography

Protein Purification and Analysis Size exclusion (gel filtration) chromatography Proteins larger than the exclusion range of the resin cannot enter the pores and so they pass quickly through the column Void volume - spaces between the resin porous beads To determine void volume load a VERY LARGE protein on column (Blue dextran, 2.000,000 Da), it will pass straight through column and give a measure of the void volume

Protein Purification and Analysis Ion exchange chromatography Separate by charge Column packed with a charged resin Use a charged buffer Like charged proteins flow through with buffer Oppositely charged proteins bind to column Elute protein Increase salt or pH to elute protein of interest

Protein Purification and Analysis Ion exchange chromatography Column- CH 2 -CH 2 -NH C2H5C2H5 C2H5C2H5 + Diethylaminoethyl (DEAE) Positively charged resin Column- CH 2 -C O O-O- Carboxymethyl (CM) Negatively charged resin

Protein Purification and Analysis Affinity chromatography Separate by specificity Column packed with: Molecules (ligands) that interact strongly with protein of interest As wash: Molecules of interest bind to column, other proteins flow through Elution: Bound proteins eluted by adding high concentration of ligand

Protein Purification and Analysis

Lab steps: Prepare gel column - “Pack a column”, create a “bed” which is the packed matrix, “bed volume” is the volume of the packed matrix (beads + void volume) DO NOT LET YOUR COLUMN GO DRY!! BE CAREFUL with CAP!!!

Protein Purification and Analysis Lab steps: Separate standards/sample application Run buffer through gel column to equilibrate column Carefully apply sample mix to top of gel bed, try not to disturb column!! Let sample drain into column and do small wash - cap column Load buffer into column and reservoir and start collecting eluant in graduated tube so you can measure volume

Protein Purification and Analysis Lab steps: Table of MW of colored molecules

Protein Purification and Analysis Lab steps: Collection of samples and measurements of Void volume (Vo) and elution volume (Ve) Ve/Vo - used in gel filtration chrom

Protein Purification and Analysis Lab steps: Determine MW of protein Estimate MW by comparing Ve of standard protein to Ve of unknown protein Create standard curve, measure Ve for unknown protein (rabbit hemoglobin)