Protein

determination
and
concentration
Protein Production for
Biophysical and Biochemical
Studies

November 2014
 Concentration
determination

Protein Quantification
 Requirements:
 Fast and easy
 Linear over a broad range
 Low cost
 Minimal interferents
 Concentration
determination

Bradford assay
 Coommassie Blue changes color from
brown to blue in presence of proteins
 Absorbance at 595 nm is measured to
quantify protein amounts
 Ratio with absorbance at 450 nm is more
exact
 Concentration
determination

Bradford assay
 Advantages:
 Simple
 No need for an additional tag / chromophore
 Compatible with reducing agents, chaotropic
and chelating agents, metals

 Disadvantages:
 A standard curve is needed (same buffer!)
 Linear over a short range of concentrations
 Dependence on MW of proteins
 Not exact!
 Interference by detergents
 Concentration
determination

Data Example
1. Measure known concentrations of BSA in duplicates
[BSA] Absorbance at Absorbance at Ratio
(µg/ml) 595nm 450nm (A595/A450)
0 0.477 0.467 0.883 0.872 0.538
10 0.603 0.599 0.843 0.829 0.719
20 0.677 0.712 0.805 0.808 0.861
30 0.795 0.782 0.758 0.770 1.032
40 0.856 0.906 0.766 0.747 1.165
50 1.004 0.971 0.715 0.754 1.346
 Concentration
determination

Data Example
1. Measure known concentrations of BSA in duplicates
[BSA] Absorbance at Absorbance at Ratio
(µg/ml) 595nm 450nm (A595/A450)
0 0.477 0.467 0.883 0.872 0.538
10 0.603 0.599 0.843 0.829 0.719
20 0.677 0.712 0.805 0.808 0.861
30 0.795 0.782 0.758 0.770 1.032
40 0.856 0.906 0.766 0.747 1.165
50 1.004 0.971 0.715 0.754 1.346

2. Compile calibration curve:

1.6
1.4
Ratio Absorbance

1.2
1.0
0.8
0.6
0.4 y = 0.0159x + 0.5471
0.2 R2 = 0.9985
0.0
0 10 20 30 40 50 60
[BSA]
 Concentration
determination

Data Example
1. Measure known concentrations of BSA in duplicates
[BSA] Absorbance at Absorbance at Ratio
(µg/ml) 595nm 450nm (A595/A450)
0 0.477 0.467 0.883 0.872 0.538
10 0.603 0.599 0.843 0.829 0.719
20 0.677 0.712 0.805 0.808 0.861
30 0.795 0.782 0.758 0.770 1.032
40 0.856 0.906 0.766 0.747 1.165
50 1.004 0.971 0.715 0.754 1.346

2. Compile calibration curve:

3. Measure protein concentration
1.6
1.4
Ratio Absorbance

1.2
1.0
0.8
0.6
Example: Absorbance ratio: 0.8
0.4 y = 0.0159x + 0.5471
0.2 R2 = 0.9985
0.0
0 10 20 30 40 50 60
[BSA]
 Concentration
determination

Data Example
1. Measure known concentrations of BSA in duplicates
[BSA] Absorbance at Absorbance at Ratio
(µg/ml) 595nm 450nm (A595/A450)
0 0.477 0.467 0.883 0.872 0.538
10 0.603 0.599 0.843 0.829 0.719
20 0.677 0.712 0.805 0.808 0.861
30 0.795 0.782 0.758 0.770 1.032
40 0.856 0.906 0.766 0.747 1.165
50 1.004 0.971 0.715 0.754 1.346

2. Compile calibration curve:

3. Measure protein concentration
1.6
1.4
Ratio Absorbance

1.2
1.0
0.8
0.6
Example: Absorbance ratio: 0.8
0.4 y = 0.0159x + 0.5471
0.2 R2 = 0.9985
0.0
0 10 20 30
[BSA]
40 50 60
4. Determine protein concentration
Concentration: ~15µg/ml
 Concentration
determination

Lowry assay
 Relieson color change in added solution
 Copper ions bind peptidic bonds (alkali
conditions)
 Aromatic residues also take part
 Concentration
determination

Lowry assay
 Advantages:
 Simple
 Compatible with detergents
 Very precise
 Compatible with many interferents after protein
precipitation with TCA (Peterson)

 Disadvantages:
 Standard curve needed
 Aromatic residues
 Lamp with near IR (750nm)
 Interference: reducing agents and others
 Concentration
determination

Absorbance at 280nm
 Aromatic side chains absorb light at
280nm
 Using Beer-Lambert law we can
determine the concentration:
A=ε x l x C
 ε= 5690 x nTrp + 1280xnTyr
 l=1cm (usually)

 Absorbance should be
between 0.1 and 1
 Concentration
determination

Absorbance at 280nm
 Advantages:
 Very precise for purified proteins
 Non-disruptive
 Additional info: pureness, aggregates

 Disadvantages:
 Need aromatic residues
 Different chromophores can interrupt
 nucleic acid, detergents, cofactors, phenolic
compounds, pigments, reducing agents, etc.
 Problematic for a mixture of proteins
 Concentration
determination

Data Example
0.45
A=ε x l x C
Absorbance

0.225

Wavelength (nm)

 Peak at 220nm cannot be quantified

 Concentration
determination

Data Example
A=ε x l x C
0.45
Abs(280nm)=0.45
C= A/(ε *l)
Absorbance

ε= 6970M-1cm-1
C= 0.45/(6970 *1)
l=1cm
0.225
C=64.5µM

280

Wavelength (nm)

 Peak at 220nm cannot be quantified

 Concentration
determination

Fluorescence
 Advantages:
 Compatible with reducing agents, detergents
and nucleic acids
 Very sensitive

 Disadvantages:
 Tag needed
 Need a fluorimeter
 Costly
 Linear only over a short range
 Calibration curve needed
 Protein
concentration

Protein concentration
methods

 Non-denaturative methods
 Denaturative methods
 Protein
concentration

Ultrafiltration
 Based on size exclusion and centrifugation
 Membrane: Polyethersulfone, cellulose triacetate
 Advantages:
 Easy use
 First choice
Protein stays on one side
 Widespread in Industry while buffer flows through

 Disadvantages:
 Slow concentration with viscous
buffers (glycerol, etc)
 Aggregation by over-concentrating
 Protein
concentration

Ultrafiltration
 Based on size exclusion and centrifugation

Examples of ultrafiltration
tubes
 Protein
concentration

The instrument
Lyophilization
 Freeze-dry samples to powder

 Resuspend in smaller volume

The process
 Protein
concentration

Lyophilization
 Advantages:
 No over-concentrating
 Keeps the protein stable
 Good choice when conditions are known

 Disadvantages:
 Conditions are hard to find
 Not suitable for all buffers (glycerol)
 Salts and additives will get concentrated too
 Takes overnight
 Dangerous for unstable proteins
 Protein
concentration

Ammonium Sulfate
Precipitation
 (NH4)2SO4 is added to protein sample
 Different proteins precipitate at different IS

 Purification: precipitating undesired proteins

 Concentrating: precipitate desired protein, then
resuspend in lower volume
 Protein
concentration

Ammonium Sulfate
Precipitation
 Advantages:
 Easy to perform
 Keeps the protein stable in most cases
 Good for concentrating proteins before GF
 Eliminates some interferents

 Disadvantages:
 Time
 High salt / needs buffer exchange

 Tip: (NH4)2SO4 takes up volume, use calculator

 Protein
concentration

Ammonium Sulfate
Precipitation
 Calculator
example:
 Protein
concentration

PEG Precipitation
 Polyethylene-glycol is a large polymer
 Generally inert, no protein interactions
 Protein will concentrate in areas of solvent
without polymer – steric exclusion
 Protein
concentration

On-Column Concentration
 Load dilute protein on column: (IEX, HIC, affinity)
 Elute up-flow at very low flow-rate in smaller
volume than loading volume

 Advantage: Efficient
 Disadvantage: Over-
concentrating, time,
availability, need of a
tag
 Protein
concentration

Denaturative concentration
methods
 Eliminate interferences before electrophoresis or protein
determination

 Proteins get irreversible denatured and lose activity!

 TCA-DOC: For precipitation of low concentration proteins

 Normal TCA: Eliminates TCA soluble interferences

 Acetone: Eliminates acetone soluble interferences

 Ethanol: Removal of GuHCl before PAGE-SDS

 Chloroform Methanol: Removal of salt and detergents

 Acidified Acetone/Methanol : Removal of acetone and methanol soluble

interferences (SDS before IEF)
Summary
 Each protein is unique!
 What suits one protein might not suit
another
 Method of choice depends on
availability, time, cost
 Be careful of over-concentrating