Protein Purification and Analysis Solubility of proteins important for purification: 60-80% soluble, 20-40% membrane Size of proteins varies Some proteins

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.)


Characteristic: Procedure:

Charge 1. Ion exchange chromatography2. Electrophoresis3. Isoelectric focusing

Size: 1. Dialysis and ultracentrifugation2. Gel electrophoresis3. Gel filtration (size exclusion) chromatography4. Ultracentrifugation

Specificity: 1. Affinity chromatography

Polarity: 1. Adsorption chromatography2. Paper chromatography3. Reverse-phase chromatography4. Hydrophobic chromatography


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


ChromatographyImportant steps in chromatography1. 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 column4. Wash column - Molecules washed through the column with buffer5. Collect fractions - Fractions are taken, at some point your molecule

will elute


Size exclusion (gel filtration) chromatographySeparate by sizeColumn packed with porous beadsAs wash with buffer:

Small molecules enter the beadsLarge 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 matrix Exclusion/fractionation rangeSephadex G-10 0.4 - 6 kD Sephadex G-25 0.8 - 20 kDSephadex G-50 1-30 kD Sephadex G-100 4-150 kD Sephadex G-200 5-600 kD Bio-Gel P-10 1.5-20 kD Bio-Gel P-30 2.4-40 kD Bio-Gel P-100 5-100 kD Bio-Gel P-300 60-400 kD

kD - kilodalton D - 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 AnalysisSize exclusion (gel filtration) chromatography


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 AnalysisIon exchange chromatography

Separate by chargeColumn packed with a charged resinUse a charged buffer• Like charged proteins flow through with buffer• Oppositely charged proteins bind to columnElute protein • Increase salt or pH to elute protein of interest

Protein Purification and AnalysisIon exchange chromatography

Column- CH2-CH2-NHC2H5

C2H5

+

Diethylaminoethyl (DEAE)Positively charged resin

Column- CH2-CO

O-

Carboxymethyl (CM)Negatively charged resin


Affinity chromatographySeparate by specificityColumn packed with: Molecules (ligands) that interact strongly with protein of interestAs wash: Molecules of interest bind to column, other proteins flow throughElution: Bound proteins eluted by adding high concentration of ligand



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!!!


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


Lab steps:

Table of MW of colored molecules


Lab steps:

Collection of samples and measurements of Void volume (Vo) and elution volume (Ve)

Ve/Vo - used in gel filtration chrom


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)

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Protein Purification and Analysis Solubility of proteins important for purification: 60-80% soluble, 20-40% membrane Size of proteins varies Some proteins