Protein Purification and Analysis

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

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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
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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 medium is an important
consideration in gel filtration Column
matrix Exclusion/fractionation range Sephadex
G-10 0.4 - 6 kD Sephadex G-25 0.8 - 20
kD Sephadex 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
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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
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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
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Protein Purification and Analysis
Ion exchange chromatography
Carboxymethyl (CM) Negatively charged resin
Diethylaminoethyl (DEAE) Positively charged resin
C2H5
O

Column- CH2-CH2-NH
Column- CH2-C
C2H5
O-
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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
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Protein Purification and Analysis
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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!!!
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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
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Protein Purification and Analysis
Lab steps Table of MW of colored molecules
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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
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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)