Download Protein Purification and Analysis Ion exchange

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:
Charge
1. Ion exchange chromatography
2. Gel 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 since early 1900s
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
Protein Purification and Analysis
Size exclusion (gel filtration) chromatography
• 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 matrix is an important
consideration in gel filtration
Column matrix
Sephadex G-10
Sephadex G-25
Sephadex G-50
Sephadex G-100
Sephadex G-200
Bio-Gel P-10
Bio-Gel P-30
Bio-Gel P-100
Bio-Gel P-300
kD - kilodalton
Exclusion/fractionation range
0.4 - 6 kD
0.8 - 20 kD
1-30 kD
4-150 kD
5-600 kD
1.5-20 kD
2.4-40 kD
5-100 kD
60-400 kD
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 Analysis
Size exclusion (gel filtration) chromatography
• 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
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
Carboxymethyl (CM)
Negatively charged resin
O
Column- CH2-C
O-
Diethylaminoethyl (DEAE)
Positively charged resin
+ C2H5
Column- CH2-CH2-NH
C2H5
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
Fast protein liquid chromatography
Separate by polarity/charge
Can use size, charge, affinity, hydrophobicity to purify
Low pressure
Protein Purification and Analysis
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:
Pack column
Run buffer through gel column to equilibrate column
Separate standards/sample application
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)