1
Lubomír Janda, Blanka Pekárová and Radka Dopitová
Kód předmětu: Bi8980
Protein expression and purification
X. Analytical methods used to study proteins
The most commonly used analytical techniques
• Determination of protein concentration
• Electrophoresis
• Enzyme assays
• Binding assays
The key characteristics of the protein:
• concentration
• purity
• relative molecular mass
• enzymatic activity
• binding properties
1. Determination of protein concentration
• Colorimetric assays (complex protein mixtures: lysates,
intermediate steps in a purification):
– Dye binding (Bradford)
– Bicinchoninic acid (Smith)
– Biuret
– Lowry
• Absorbance assays (purified proteins):
– absorbance at 280 nm
– absorbance at 205 nm
1.1. Colorimetric assays – general approach
• Comparative, i.e. based on the use of a reference (standard) protein
– Preparation of stock solution of reference protein (BSA, IgG)
– Construction of calibration curve for reference protein
– Determination of unknown protein using the calibration curve
• Work within the linear range of an assay, that is, where absorbance is
directly proportional to concentration.
• When samples are so concentrated that you cannot pipette a small
enough amount accurately, you may have to conduct serial dilutions.
1.1.1. Dye binding assay – Bradford
Principle:
The absorbance maximum (λmax) for an acidic solution of Coomassie
Brilliant Blue G-250 shifts from 465 nm to 595 nm when binding to
protein occurs.
– λ max 465 nm: reddish/brown (protonated form)
– λmax 595-620 nm: blue (deprotonated form)
• dependent on the amino acid composition – the dye reagent reacts with
side chain NH3
+ groups → primarily with Arg and less so with Lys, His,
Tyr, Trp, and Phe
• is less accurate for basic or acidic proteins
• free amino acids, peptides and low molecular weight proteins (<3,000
Da) do not produce color with Coomassie dye reagents
Bradford, M. M. (1976): Anal. Biochem. 72: 248-254
1.1.1. Dye binding assay - Bradford
Advantages
– fast and inexpensive
– highly specific for protein
– very sensitive: 1–25 µg
– Compatible with a wide range of substances (major interfering agents:
detergents (SDS))
Disadvantages
– assay is linear over a short range
– response to different proteins can vary widely;
choice of standard is very important
Modification:Thermo Scientific Pierce 660 nm Protein Assay – compatible with
most detergents and produces a more linear response curve
1.1.2. Bicinchoninic acid (BCA or Smith) assay
Principle
Under alkaline conditions, Cu2+ present in the BCA reagent forms
complexes with peptide bonds and becomes reduced to Cu+. Cu+
subsequently interacts with BCA to form a purple BCA-Cu+ complex
detectable at 562 nm.
Smith et al. (1985) Anal. Biochem. 150: 76-85.
green → purple
1.1.2. Bicinchoninic acid (BCA or Smith)
assay
Advantages
– very sensitive 0.2–50 µg
– compatible with many detergents
– broad linear working range
– little variation in response between different proteins
Disadvantages
– long incubation time (~1 h), required warming (60°C) → This
can be a problem if it is assaying a large number of proteins.
– major interfering agents: glucose, strong acids (EDTA),
ammonium sulfate, lipids
1.1.3. Biuret assay
Principle
Under strongly alkaline conditions proteins containing two or more
peptide bonds form a purple complex with Cu2+ salts in the Biuret
reagent (absorption at 540 nm).
Advantages
- very little variation in response between different proteins
- broad linear working range
Disadvantages
- low sensitivity: 0.5–5 (10) mg
- major interfering agents: ammonium salts
1.1.4. Lowry assay
Principle
Based on two different reactions:
1. The formation of a Cu2+ complex with peptide bonds, forming
reduced Cu+ in alkaline solutions (biuret reaction).
2. The reduction of Folin-Ciocalteu reagent (phosphomolybdictungstic
acid) by Cu+, together with side chains of Tyr, Trp, Cys,
and His.
The reduced Folin-Ciocalteu reagent is blue → detectable at 750 nm.
Advantages
– sensitive: 5–100 µg
– the standard curve is linear
– very little variation in response between
different proteins
Lowry et al. (1951) J. Biol. Chem. 193: 265.
1.1.4. Lowry assay
Disadvantages
– many interfering agents: detergents, carbohydrates, glycerol, DTT,
tricine, EDTA, 2-mercaptoethanol, Tris, potassium compounds,
sulfhydryl compounds, disulfide compounds, magnesium and calcium,
Tyr, Trp, Cys, His, and Asp
– takes 40 min
– alkaline copper reagent must be prepared fresh daily
Modification of Lowry
• The Bio-Rad DC (detergent compatible) protein assay
• The Bio-Rad RC DC protein assay
– For protein determination in the presence of reducing agents and detergents.
• Hartree-Lowry: Hartree (1972), Anal. Biochem. 48: 422-427.
– This modification makes the assay linear over a larger range than the original
assay.
• J.R. Dulley and P.A. Grieve (1975) Anal. Biochem. 64: 136.
– This modification includes 0.5% sodium dodecyl sulfate in the alkaline reagent.
This obviates interference from many detergents and helps disperse membranes
in the sample.
• A. Bensadoun and D. Weinstein (1976) Anal. Biochem. 70: 241.
– Modification that can be useful when the solution contains interfering
contaminants. The proteins in the samples are precipitated by a mixture of
sodium deoxycholate and trichloroacetic acid and centrifugation prior to assay. If
the contaminants stay in the supernatant they can be removed and the amount
of precipitated protein determined.
1.2. Absorbance at 280 and 205
• For pure proteins
• Fast and convenient, no additional reagents or incubations are required.
• No protein standard needs to be prepared.
• Buffer must not absorb at 280/205, or, if it does, an accurate blank must be
taken (imidazole, HEPES, MOPS absorb in the far UV).
• Non-aggregated proteins (presence of aggregated proteins leads to light
scattering, increasing absorbance values)
• Significant levels of nucleic or oligonucleotide fragments → A205
A280/A260 for proteins ~ 1.7
(for nucleic acids ~ 0.62)
1.2.1. Absorbance at 280 nm
• Radiation absorbance due to Tyr, Trp and, to a lesser extent, Cys
• A280 → protein concentration: requires the molar absorbance coefficient (ε)
determined from amino acid composition
Denaturated (unfolded) protein:
ε of the protein (280 nm: M-1cm-1)=(∑ Tyr x 1280)+(∑ Trp x 5690)+(∑ Cys x 60)
(modified from Gill & Hippel, 1989)
Native (folded) protein:
ε of the protein (280 nm: M-1cm-1)=(∑ Tyr x 1490)+(∑ Trp x 5500)+(∑ Cys x 62.50)
(Pace et al., 1995)
[in both equations all Cys form disulfide bonds (cystines), if not: ∑ Cys = 0]
1M solution of protein, of molecular mass X Da, is X mg/ml:
For 1 mg ml-1 protein solution: A280 = ε of the protein / Mr (Da)
(A280 for 1 mg ml-1 protein solution, calculated according to equation 1, are available from
Protparam (Expasy), Swiss-Prot, TrEMBL)
A280 between 0.1 and 1
1.2.2. Absorbance at 205 nm
• Peptide bonds absorb radiation < 240 nm
• For proteins and peptides with low Tyr, Trp
• For proteins with significant nucleic acid contamination
• Protein c (mg mL-1) = A205/31
• But more reliable: A205(1mg mL-1) = 27+120 (A280/A205) (Scopes, 1974)
– factor 27: A205 of 1mg mL-1 protein solution
– factor 120: empirical value (chosen from studies of variety of standard
proteins) to take into account the contribution of Tyr and Trp at 205 nm
2. Electrophoresis methods
• One-dimensional SDS-PAGE (denaturating PAGE)
• Native PAGE (non-denaturating PAGE)
• Isoelectric focusing
• Two-dimensional gel electrophoresis
• Immunoelectrophoresis
2.1. One-dimensional SDS-PAGE
= sodium dodecyl sulphate-polyacrylamide gel electrophoresis
Applications: - estimation of protein subunit molecular mass
- assessment of the protein purity
Proteins are separated according to
their size – SDS → constant negative
charge: for most proteins
1.4 g of SDS/1 g of protein
!Membrane proteins – gel shift!
Rath et al., 2009
CKI1 170
Wt Ox2-2 M
130
95
72
kDa
CKI1: 125 kDa
163 kDa
2.1. One-dimensional SDS-PAGE
Discontinuous system – Laemmli (1970)
10-4312-6020-8036-9457-212Separation
range (kDa)
1512107.55%
Acrlyamide
Effective separation range of uniform polyacrylamide gels
2.1. One-dimensional SDS-PAGE
Experimental design
Scale of analysis
- small format (8 x 10 cm): rapid separation
- large format (20 x 16 cm): when high resolution is required
Complexity of the sample
- sample of a few proteins: 1 µg/lane (small format, CB staining)
- complex sample: 20 µg/lane (small format, CB staining)
Suitable detection methods
- Coomassie Brilliant Blue: 0.5 ug
- silver: 0.1ug
- immunodetection: ng
Percentage of polyacrylamide gel
Type of marker
- mobility greater than and less than protein of interest
- mobility in a linear fashion relative to log (molecular mass)
2.2. Native electrophoresis
• Proteins are separated by their size, charge and shape
• Non-denaturing conditions → proteins remain stable and in their native form
→ structure and function retention
β-glucosidase
• Determination of molecular mass, charge, subunit composition (quaternary
structure)
– Calibration proteins with similar mass,
different charge → protein charge determination
– Calibration proteins with similar charge,
different mass → protein mass determination → Ferguson plot (evaluated
using program ElphoFit)
4-methylumbelliferyl-β-D-glukopyranosid (MUG)
β-glucosidase
4-methylumbelliferon β-glukosa
2.3. Isoelectric focusing (IEF)
It is a high-resolution method which separates proteins according to their pI.
Proteins that are structurally very similar and have pI that differ by as little as
0.1 pH units can be resolved by IEF.
Proteins carrying a net positive charge migrate towards the
cathode and lose or gain protons (if below or above, respectively)
until they reach pH=pI
Proteins carrying a net negative charge migrate towards the
anode and lose or gain protons (if below or above, respectively)
Success relies on the ability to resolve the proteins in the
sample → optimization of pH gradient range and the size
of electric field
Proteins in the sample must be soluble and free from salts
http://www.bio.miami.edu/~cmallery/255/255tech/ecbxp4x4_focus.jpg
2.4. Two-dimensional gel electrophoresis
Proteins are separated in two dimensions:
IEF → SDS-PAGE
Applications:
– analysis of complicated mixtures
– quantification of each protein within a complex mixture
– identification of changes in protein expression pattern
– characterization of subtle post-translational modifications
Experimental design
– loading of the optimal amount of sample to allow separation and detection of
proteins which are abundant as well as those which are less abundant
– selection of a suitable pH range for the first dimension
– selection of an appropriate acrylamide gel for optimal separation in the second
dimension
– staining: silver (0.1 µg)
Fluorescent detection: Sypro Ruby (1–2 ng), Sypro Rose (1–2 ng),
Flamingo
Radioactive detection
2.5. Immunoelectrophoresis
Antigens (Ag) and antibodies (Ab) are separated by migration through an electric
field
Specific Ag-Ab interactions → formation of precipitates (precipitin)
In low concentration agarose gels (1%), Ag migrate towards the anode, Ab move
towards the cathode.
Techniques:
• Cross-over electrophoresis
Ab and Ag are added to the wells.
• Quantitative immunoelectrophoresis (rocket immunoelectrophoresis)
Ab is added to the agarose during gel preparation, Ag in the wells.
• Two-dimensional immunoelectrophoresis
Ag are separated by native elfo and subsequent gel is
applied to the top of an agarose gel impregnated with Ab.
Application of electric field → migration of Ag into the agarose
gel → precipitin
+
-
3. Enzyme assays
Enzyme assays = methods by which we can obtain quantitative information
about the catalytic activity, including parameters such as the Michaelis
constant (Km), maximum (or limiting) velocity (Vmax, kcat), and the catalytic
efficiency kcat/Km.
By studying the activity under different conditions in vitro, we can evaluate
how the enzyme works under conditions close to those operating in vivo
→ this may help in the design of specific inhibitors (simvastatin = HMGCoA
reductase inhibitor treats high blood cholesterol levels).
The normal approach to study enzyme activity is “steady-state” conditions:
the concentrations of enzyme-substrate complexes remain essentially
constant. The enzyme is present at a very small concentration: 0.1% or
less of the substrate concentration.
Enzyme assays
3.1. Continuous
3.2. Stopped (Discontinuous)
3.1. Continuous assays
The conversion of substrate to product can be continuously observed.
Spectrophotometric (absorbance)
Most dehydrogenases used NAD(P)+/NAD(P)H+H+ redox system
NAD(P)H absorbs radiation at 340 nm, NAD(P)+ does not
Alcohol dehydrogenase
Ethanol + NAD+ ↔ acetaldehyde + NADH + (H+)
Fluorometric (fluorescence - molecule emits light of one wavelength after
absorbing light of a different wavelength)
Calorimetric (calorimetry is the measurement of the heat released or
absorbed by chemical reactions)
Chemiluminescent (chemiluminescence is the emission of light by a
chemical reaction)
horseradish peroxidase + TMB substrate (ELISA)
3.1.1. Coupled assays
When the enzyme-catalyzed reaction of interest does not lead to any
convenient spectroscopic signal, it is possible to couple this reaction to a
second one in which such a change occurs.
Hexokinase
D-glucose + ATP→ D-glucose-6-phosphate + NADP+
Glucose-6-phosphate dehydrogenase
D-glucose-6-phosphate + NADP+→ D-glucono-1,5-lactone-6-phosphate + NADPH + H+
It is important that:
– a considerable excess of coupling enzyme (50-times greater) be added,
– the coupling substrate be at a concentration above Km for the coupling
enzyme, and
– the coupling enzyme be free of any detectable amount of the enzyme
being assayed.
3.2.2. Stopped (discontinuous) assay
Enzyme is incubated with substrate for a fixed period of time
Before analysis:
• inhibition of the enzyme
– rapid changes in pH
– rapid heating (90 °C sufficient to denaturate most enzymes)
– addition of trichloroacetic or perchloric acid (to final c = 3–5%) –
neutralization before product measurement
– addition of SDS to 1%
• establish that:
– the enzyme is instantaneously inactivated
– conditions used do not lead to any breakdown of the substrate or product
being measured
Radiometric assays measure the incorporation of radioactive isotopes (14C, 32P,
35S and 125I) into substrates or its release from substrates.
Chromatographic assays measure product formation by separating the reaction
mixture into its components by chromatography (HPLC, RP-HPLC)
Chemical measurement (for example: phosphate + non-precipitating acid
molybdate → color formation)
How to ensure that valid kinetic data is
obtained
1. It is important to use buffer components and substrates of high
purity (analytical grade).
2. A control experiment should be performed in the absence of
enzyme.
3. Experimental variables (temperature, pH) are controlled.
4. Enzyme is stable under the conditions used for assay.
5. The initial rate should be measured.
6. The rate of the reaction is constant over the time period used to
measure the rate, and this rate is proportional to the amount of
the enzyme added.
4. Binding assays
Proteins bind other molecules (ligands): proteins, nucleic acids,
carbohydrates, or small molecules
Quantitative analysis:
• What is the strength of the protein-ligand interaction?
• How many binding sites for the ligand are there on the protein?
• If there are multiple ligand binding sites, are they independent or are
there cooperative interactions between them?
• Is the strength of the protein-ligand interactions modified by other
molecules?
4.1. Methods involving separation of the protein-ligand complex
(on the basic of size)
4.2. Methods involving a signal change on complex formation
4.3. Competition methods
4.4. Immunochemical methods
4.1. Methods involving separation of the
protein-ligand complex
4.1.1. Equilibrium dialysis
• At equilibrium: inside [Lt] = [Lb] + [Lf], outside [Lt] = [Lf]
→ measurement of L, PL, P concentrations
• Applications
– to study the binding of ligands with low molecular mass
4.1.2. Electrophoretic mobility shift assay (EMSA)
• Widely used technique for studying the binding of proteins to DNA and
RNA (or their fragments)
• Based on: The mobility of the nucleic acid upon electrophoresis in an
agarose or polyacrylamide gel is reduced when it forms a complex with
the appropriate protein.
• Can be used to monitor other types
of interaction (P-P).
t: total, b: bound, f: free
4.1. Methods involving separation of the
protein-ligand complex
4.1.3. Ultracentrifugation
In gravitational fields, proteins have a tendency to sediment at a rate which
depends on their size and shape.
Approaches
• Measuring the concentration of small ligands (difference in L sedimentation
rate (L alone and L+P) to detect its binding to protein).
• Centrifuging the mixture of P and L at a high speed for a sufficiently long
time to sediment the P and PL complex and then analyze the protein-free
supernatant for the free L.
• Exploring the interaction between P and a high molecular mass L (another
P). Solution is centrifuged at moderate speed for several hours so that
equilibrium is established between the tendency of the macromolecules to
sediment to the bottom of the cell and their tendency to diffuse away from
the bottom of the cell to a region of lower concentration.
4.2. Methods involving a signal change on
complex formation
• Changes in fluorescence
– (fluorescent ligands, fused proteins with GFP, YFP,..)
• Circular dichroism (CD)
– CD depends on the differential absorption of the left- and right-circularly polarized light
by a chiral chromophore. Changes in the CD spectra are directly proportional to the
amount of the protein-ligand complexes formed.
• Nuclear magnetic resonance
– The spectroscopic properties of each nucleus are sensitive to its chemical
environment → can be used to monitor protein–ligand interactions and characterize
them in thermodynamic terms. NMR provide great structural information about the
amino acid residues in the protein that interact with the ligand.
• Isothermal titration calorimetry (ITC)
– directly determines changes in thermodynamic parameters (∆H, ∆G, ∆S)
• Surface plasmon resonance
– Based on changes of the optical properties of a surface layer containing immobilized
protein with increasing mass of protein bound from mobile fluid phase.
4.3. Competition methods
When a ligand does not show an appropriate signal change (for example,
fluorescence) upon binding → competition of the ligand with a reference one
of the same binding site that shows such change.
4.4. Immunochemical assays
Based on the specific binding of an antibody (Ab) to an antigen (An).
4.4.1. Enzyme-linked immunosorbent assay (ELISA)
• Ag and Ab detection and quantification
• Detection and quantification of
protein-protein interactions
Daussant & Desvaux, 2007
4.4.2. Western blot
After gel electrophoresis proteins are transferred onto a membrane, they are
detected using specific antibodies.
Membrane: NC, PVDF
Blocking agent: non-fat milk,
BSA
Enzyme: alkaline phosphatase
horse radish peroxidase
Detection: colorimetric or
chemiluminescent
Blot overlay (Far Western blot)
To study protein-protein
interactions, proteins transferred to
membrane are overlaid with a
soluble protein that bind to one or
more immobilized proteins.
Overlaid protein is detected via
incubation with antibodies.
Electrotransfer
1. Semi-dry
has lower buffering capacity and thus is not suitable for prolonged transfer
2. Wet tank transfer
Towbin transfer buffer, pH 8.3
If a protein has pI equal to the buffer pH, transfer will not be promoted.
Higher pH buffers such as CAPS or lower pH buffers such as acetic acid
solutions can be used.
High Mw proteins or membrane proteins may be very hydrophobic and
exhibit solubility problems in the absence of SDS → SDS in buffer < 0.05%.
Transfer efficiency: reversible Ponceau S red staining of membrane and/or gel
staining
4.4.3. Immunoprecipitation
Isolation of antigen (protein) from a non-homogeneous solution, such as a
cell lysate, using specific antibodies. Ag-Ab complex is captured by Abbinding
proteins (like protein A or G), which should
only bind the antibody and whatever is
associated with it. The bound Ag-Ab
complex is subsequently released from
the Ab-binding molecule and analyzed
by SDS-PAGE and Western blot.
Applications
• to determine the presence and quantity
of proteins
• to determine protein molecular mass
• to determine protein-protein interactions
• to determine specific enzymatic activity