Download Electrophoresis Chapter 10 +

Electrophoresis
Chapter 10
ARNE TISELIUS
Nobelprize in Chemistry 1948
Motivation:
“for his research on electrophoresis
and adsorption analysis, especially
for his discoveries concerning the
complex nature of the serum proteins"
Electrophoresis can only be performed
on molecules having a charge.
+
- ++ ++- +
++
- + ++- ++
+
-
+
++ ++
++ ++ +
+
++
+
--- -
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Good things with electrophoresis
• Relatively simple and inexpensive
equipment
• High resolution results
• Easy to analyze multiple samples
• High sensitivity
• Specific detection easy (immunological,
enzymatic)
Different formats
• Tubegels: were first used
• Vertical slab-gels: most common when
separating proteins.
• Horizontal slab-gels: IEF and 2D-gels
• Capillary electrophoresis
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Formulas
V =E
d
V = voltage
d = distance
E = Potential gradient
v = Eq
f
v = velocity
q = charge
f = friction coefficient
v
µ= E
µ = eletrophoretic mobility
Formulas
V = I*R
W =I2R
R = Resistance
I = Current
V = Voltage
W = Effect
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Heat is generated
1. Increased diffusion -> band broadening
2. Convection currents -> Mixing of sample
3. Heat-sensitive proteins can be damaged.
4. Lower viscosity of the buffer lead so a
reduction of the friction coefficient -> faster
velocity of the sample.
Electroendosmosis
- liquid movement (in the capillary)
- - - - - - - - - - - - +
+
+
+ + +
+
+
- - ++ ++ +
++ - +
++
+
++
+ ++
- +
++
++
++
++
+
-
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Supporting media (1)
avoid e.g. convection currents
Agarose
•
•
•
•
Polysaccharide (same as in agar)
Both intra- and inter-molecular binding
1-3% gels are used
Used to separate proteins and DNA. Somewhat large
pores for proteins.
• Uncharged. However, substitutions may occur
(avoid electroendosmosis)
• The pores are suitable for size separation of RNA and
DNA
Supporting media (2)
avoid e.g. convection currents
Polyacrylamide PAGE (Poly Acrylamide Gel
Electrophoresis)
• Polymerization of acrylamide and bis-acrylamide, the amount of
bis determines the decree of cross-coupling
• Catalysis by a free radical: R* +M -> RM*
RM* + M -> RMM* etc.
9degas, Oxygen can react with the free radical or create
airbubbles in the gel
9Photopolymerization: Riboflavin
9Chemical polymerization : TEMED and
ammoniumpersulphate
• Depending on the desired frictional constant, 3 to 30% gels are
used.
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SDS-PAGE
Sodium-Dodecyl-Sulphate PAGE
Separation by size
Determination of relative size -> compare with a molecular size
marker
Control of purity between and after a purification step.
Sample:
• Boiled with SDS, a detergent CH3-(CH2)10-CH2OSO3-Na+
binding to the protein and denaturating it (1SDS/2 aminosyror)
-> all proteins becomes negatively charged, the amount of
charge is reflecting the size.
• A reducing agent is used to reduce all disulphide bridges.
• A color in the sample is reflecting how far the sample has moved,
BromphenolBlue (BFB)
• Glycerol to increase the density of the sample.
Stacking gel
The topmost part of the gel where the sample is concentrated
to a narrow band
• Large pores ( ≈4%) -> low friction, size independent
• Not the same buffer, lower pH than the separation gel
• Different ion have different mobility:
Glycinate< Proteins with SDS <ClMust move with the same velocity – a higher field-strength is
needed. This is compensated by a concentration of the
sample.
[Cl-]>[SDS-proteines]>[glycinate]
• When reaching the separation gel, the pH goes up and glycinate
increases its mobility and moves faster than the SDS-proteincomplexes.
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Separation gel
The proteins are separated according to size.
•The same charge/length results in a mobility which is
reflecting the size.
• The network in the gel is enhancing this effect,
friction.
Higher portion of acrylamide for smaller proteins.
•The color, BFB, is moving in the front of the sample.
•15% gel is separating ≈10-100 kDa
Example gel
M
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Native PAGE
• Non-denaturating conditions – the activity of the protein is
retained.
• The proteins are separated according to size (the network of the
gel) and charge
Is the protein moving in the right direction?
• High resolution
• Difficult to predict the behavior of the protein.
• Staining for activity
Gradient gels
PAGE
• The concentration of acrylamide is varying in the gel, lowest
concentration in top of the gel and highest in the bottom.
• Is made using a gradient mixer.
• Is commonly used in combination with SDS and a stacking gel.
• Gives a larger size interval for the analysis.
• Proteins of approximately the same size are easier to separate.
• The bands get sharper, “pore-size limited”
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Isoelectric focusing, IEF (1)
• Separation by pI
• The gel contains a pH-gradient
• High resolution, down to 0.01 pH-unit
• Often horisontal gels
• The sample can be loaded anywhere on the gel.
• High voltage (>2500V) leads to generation of heat,
cooling is needed.
• Micro-heterogeneities in the sample can be analyzed.
Isoelectric focusing, IEF (2)
• Large pore-size to avoid effects by differences in size of
the proteins.
Often PAGE, ≈4%, sometimes even agarose for
large proteins.
• The gel is polymerized as usual but ampholytic
molecules are mixed with the acrylamide.
A voltage is applied to the gel which leads to a pH
gradient.
The sample is loaded and separation is performed.
When the protein has reached a pH in the gel
where its net-charge is =0 it will stop.
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Example of an IEF-gel
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2
3
4
5
6
7
5.85
5.20
4.55
4.15
2D-gel electrophoresis (1)
combination of IEF (pI) and SDS-PAGE (size)
•The first dimension, IEF, is carried out in a rod-shaped
gel, d=1-2 mm.
•8M Urea and non-ionic detergent.
•The gel is incubated with an SDS-containing buffer.
•Separation in the second dimension is performed by
putting the strip on top of the stacking gel.
•High resolution 1000-2000 (10000) proteines can be
separated
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2D-gel electrophoresis (2)
Combination of IEF (pI) and SDS-PAGE (size)
pI
size
Proteome analysis
• The genome gives an indication of the different
proteins that can be expressed in a cell. (≈22000
unique proteins in humans)
• The proteome gives an indication of which proteins
are expressed in a particular cell-type and to what
extent they are expressed at a certain point in time.
• Post-translational modifications are important for
the function of proteins and can not be deduced
with certainty from the DNA-sequence.
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2-D gels and proteome analysis
1.
2.
3.
4.
5.
Separation of proteines by 2dimensional gel elektrophoresis
Elution of the proteins from the
gel
Trypsin digestion
Masspectrometry
Data-base analysis
Or:
1. Elution of the proteins from the gel
2. Mass spectrometry
3. Data-base analysis
Staining and analysis of the gel
z
z
z
z
Most commonly by “Coomassie Brilliant Blue” (0.1% w/v)
mixed with Methanol (Ethanol), H2O, Acetic acid.
More sensitive is Silver staining, silver-ions are reduced to silver
on the protein (Limit of detection is approx 1 ng protein bands)
Glycoproteines can be stained with periodic acid-Schiff (PAS)
The amount of protein in the bands can be estimated by using a
scanning densitometer. It measures the intensity of the bands by
scanning with a laser-beam and measuring transmitted light.
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Western blotting (1)
protein blotting
Capillary blotting: Capillary forces are used to transfer the
proteines to a membrane:
Something heavy
Dry membrane
Nitro-cellulose filter, equilibrated in buffer
Gel
Electroblotting: A current is used to transfer the proteins to a
membrane:
+
-
Nitro-cellulose filter, equilibrated in buffer
Gel
Western blotting (2)
protein blotting
ÐBlocking of the nitro-cellulose filter
ÐAffinity ligand binding the target protein is applied, usually
an antibody
ÐSecondary antibody that is labeled is applied, usually with a
covalently attached enzyme.
ÐA substrate for the enzyme is added. The product should
form a colored insoluble precipitate.
Enzyme
Secondary antibody
Primary antibody
Target protein
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Electrophoresis of DNA
• Usually an agarose gel, separation by friction, pore-size of the gel.
• ´The separated bands can be compared to a marker with DNA pieces of
known size.
For dsDNA the unit kb (kilobases) is used
For ssDNA the unit nt (nucleotides) is used
• Electrophoresis is performed totally submerged in buffer (like a
submarine).
• Glycerol and BFB in the samples
• Stacking gel is not needed since the migration through the gel is very
slow.
• Staining with Ethidium bromide, visible in UV-light
• Limit of detection approx 10 ng.
DNA sequencing in
Polyacrylamide gels
• Separation of small ssDNA fragments (up
to approx 1000 nt)
• DNA sequencing is performed in PAA-gels
since fragments differing in size by only
one base can be separated.
• DNA sequencing was previously performed
in slabgels. Now it is usually being
performed in cappilaries.
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Electrophoresis of large DNA
molecules
• Pulsed-field Electrophoresis, PGFE, is used to separate very large
DNA-molecules, 2*103 kb
• The angle of the electric field is changed every 60 seconds.
Electrophoresis of RNA
Northern blotting
Adapted from
Professor Roger L. Miesfeld
The University of Arizona
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