C8116 Immunoaffinity techniques
Advanced microscopy
Spring term 2024
Hans Gorris
Department of Biochemistry
April 30th, 2024
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Time-resolved fluorescence
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Sequential absorption of 2 or more photons via long-lived transition states
=> More time for absorbing a further photon 3
Photon-upconversion
no PSA 10 fg/mL 100 fg/mL
1 pg/mL 10 pg/mL 100 pg/mL
1 ng/mL 10 ng/mL 100 ng/mL
Anal. Chem. (2017) 89, 11825
4
Counting single immune complexes
non-specific binding only
=> Detectable as diffraction limited spots
5
Digital immunoassay allow for the detection of single analyte molecules,
but this should not be confused with the highest analytical sensitivity
=> non-specific binding of labeled component (and
its variation) defines the actual limit of detection
non-
specific
specific
Single molecule (digital) assays
6
“Smart“ reporters for
heterogeneous immunoassays
“Smart“ reporters
7
How to avoid signal from nonspecific binding?
solid phase
no signal no signal
“Smart“ reporters
8
solid phase
analyte
”smart” reporter gives signal only
when both parts come together
Modulation of high specific activity signal upon recognition of analyte
no signal no signal
Fredriksson (2002): Protein detection using proximity-dependent DNA ligation assays. Nat. Biotechnol. 20: 473-477
Proximity ligation
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Homodimeric PDGF-BB
Proximity probe
A1
Proximity probe
A2
Aptamer specific
for PDGF-B
Aptamer specific
for PDGF-B
primer primer
connector oligonucelotide
probe for detection
of PCR products
F: fluorescent dye fluorescein
Q: quencher TAMRA
sandwich immunoassay with two DNA-labeled detection
antibodies and one solid-phase bound capture antibody
amplified
by PCR
0.001 % 0.001 %
3'P-5'
5'
3' 5' 3' 5'
3'
3' 3'
5'-P
3'5'
Proximity ligation in immunoassays
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Proximity ligation in immunoassays
only specific binding is detected
- to produce signal, two labeled antibodies need to simultaneously bind to different
epitopes of the same analyte
- non-specifically bound individual antibodies do not produce any signal
=> highly sensitive technology for protein detection
but: complex to perform, in total 3 antibodies against different epitopes are required
11
=> enables more sensitive detection than other assays due to
high specificity in signal generation
Signal(au)
Analyte concentration (M)
0
10
100
1000
10000
100000
0 10-1410-19
1000000
10-17 10-16 10-15 10-1310-18 10-12
Proxim
ity
ligation
assay
(PLA)Detectionlimit
Detectionlimit
Proximity ligation in immunoassays
12
=> one of the next projects in our lab
Digital immunoassays: two-color colocalization
13
solid phase
=> detection of 2 non-interacting reporters
non-specific binding:
only one color per spot
non-specific binding:
only one color per spot
analyte
specific binding:
two colors in one spot
14
Immunoblotting
=> Antibodies for the detection of
proteins immobilized on a membrane
Recapitulation: Immune precipitating systems
antigen
antibody
1% agarose
1-dimensional diffusion
2-dimensional diffusion
A) Oudin
B) Oakley / Fulthorpe
C) Mancini
D) Ouchterlony
simple diffusion
double diffusion
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Simple form: dot blot
Peroxidase-antiperoxidase method (PAP)
=> Increases the amount of enzyme / signal strength
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Example of epitope mapping: Detection scheme:
β2-microglobulin (β2-m, 0.05 mg/mL)
are spotted on a nitrocellulose membrane:
(1) amino acids 1-99 (intact peptide)
(2) amino acids 1-19 (fragment)
(3) amino acids 9-24 (fragment)
(4) amino acids 20-36 (fragment)
=> Various antibodies (B-F) bind to different parts of β2-m
Western blotting
Northern Blot
(RNA)
Southern Blot
(DNA)
Western Blot
(proteins)
Transfer ("blot") of proteins to a membrane
=> typically nitrocellulose or PVDF
(high non-specific binding of proteins;
blocking required before detection reagents can be applied)
Then protein detection
- via (labeled) antibodies specific for the target protein
- by mass spectrometry
- proteolytic degradation for sequencing
graphite plate
graphite plate
filter paper with buffer
gel
membrane
filter paper with buffer
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Western blotting
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Western blot detection
Fluorescent detection: Host cell protein (HCP) analysis
Fluorescent total
protein pattern
HCP-specific immunostaining,
detection by Cy3-secondary
antibody conjugate
Overlay
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Analysis of proteinprotein
interactions
A) In vitro
=> protein interactions investigated in a test tube
B) In vivo
=> protein interactions investigated in living organisms
“bait“
(target molecule)
“prey“
(interacting partner)
2 interacting proteins
Analysis of protein-protein interactions
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Proteins blotted on a membrane (bait) are incubated with interacting proteins (prey)
Far Western Blotting
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(1) potential interaction partners are
transferred from the gel to a membrane
(2) tagged protein is added
(3) HRP-coupled secondary antibody
binds to protein tag
Chemiluminescent signal
Co-immunoprecipitation
23=> e.g. use of protein A coated beads for immobilizing antibodies
Affinity chromatography
One binding partner (here: insulin) is immobilized on solid support (bead), the other
(the "analyte"; here the insulin receptor) is contained in the (usually complex) sample.
1. Receptor (red) specifically binds to ligand (green) when passing the column
2. Bound receptor is then washed off with a chaotropic reagent or with acid
1. Biospecific / biomimetic binding pairs:
- ligand / receptor
- antibody / hapten The column is usually
- substrate / enzyme covalently modified with
- single stranded DNA the first binding partner.
- lectin / carbohydrate
2. Metal chelate
- His-tag Requires recombinant protein
Binding constant (KD) should be 10-5 - 10-7 M
e.g. biotin-streptavidin (KD = 10-14 M) less suitable => almost irreversible binding
=> highest selectivity compared to other types of chromatography
=> high capacity for target protein
But: - more knowledge about target protein required
- longer preparation time
Variations of affinity chromatography
Preparation of recombinant proteins
N
H
CH C
CH2
O
HN
N
H
N CH C
CH2
O
N
NH
H
N CH C
CH2
O
HN
N
H
N CH C
CH2
O
N
NH
H
N CH C
CH2
O
HN
N
H
N CH C
CH2
OH
O
N
NH
N
O-
O-
O
Ni2+
O-
O
O
N
O-
O-
O
Ni2+
O-
O
O
N
O-
O-
O
Ni2+
O-
O
O
=> Insert six times the codon CAT or CAC after the DNA sequence for the protein
Washed off from the column
by a small molecule competitor
imidazole
NHN
solid phase
protein
Protein purification: His6 tag
Protein-protein interactions: GST pulldown assay
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prey protein (carries a
radioactive label, e.g. 35S)
bait protein
glutathion S-transferase
glutathion
sepharose
beads
non-bound
proteins
Surface structure of glutathione beadsAffinity chromatography
GST pulldown assay: co-binding
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Recombinant DNA techniques are
used to make fusion between protein
X and glutathione S-transferase
GST pulldown assay: gel and autoradiogram
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GST fusion PX domain of…
Size
marker
2x affinity
chromatography
Multi-subunit
complexes can
be isolated
Tandem Affinity Purification: TAP tagging
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=> 2 washing steps, less non-specific binding, milder conditions
Protein complexes analyzed by MS
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Separation and detection of protein-protein complexes
Svedberg equation:
v = [d2 (rp – rm) g]/18h
where v = rate of sedimentation
d = diameter of particle
(rp – rm) = difference in the density
of particle and medium (water/sucrose)
g = gravity applied (routinely at 10,000;
ultracentrifugation: 150,000)
h = viscosity of medium
Note: g depends on the rotational speed
and the rotor diameter!
Analytical ultracentrifugation
Detection:
UV absorption and/or interference optical refractive index
through two windows of quartz glass in the rotor
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Method Advantages Disadvantages
Far Western
blotting
• Both cloning and
heterologous expression and
detection by specific
antibodies is possibly if
antibody is available
Co-immuno-
precipitation
• Does not require cloning and
heterologous expression
• Rapid if antibody is available
• Not generic: requires access to
specific antibodies
Affinity pulldown • Generic ability to purify lowabundance
protein
complexes
• The presence of a protein tag may
influence results
• Competition with the endogenous
complex
Tandem affinity
purification (TAP)
• Generic ability to purify lowabundance
protein
complexes
• Mild conditions used
throughout
• The presence of a protein tag may
influence results
• Competition with the endogenous
complex
Analytical
ultracentrifugation
• Does not require cloning and
heterologous expression
• Rapid if antibody is available
• Expensive equipment required
Analysis of protein-protein interactions in vitro
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Yeast 2-hybrid
system (Y2H)
In vivo
Þ Protein-protein interactions are investigated in their natural environment
Y2H: Protein fragment complementation assay
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Þ remember:
“smart reporters”
Detection of reporter gene expression
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5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal)
(no color)
5,5‘-dibromo-4,4‘-dichloro-3-indigo
(blue)
DNA-binding domain
Vector for coding bait fusion protein
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Polylinker region:
Insertion of cDNA library
activator domain
Vector for coding prey fusion protein
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Yeast two-hybrid system
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(DNA binding) (activator)
growth blue staining
bait prey
Large libraries of cDNA can be screened for protein interactions
in their natural environment
But many false negative and false positive results (up to 70 %)
- Fusion proteins are overexpressed
- Proteins that are loacted in different cellular compartments interact
- Fusion proteins may inhibit interactions
- Posttranslational modifications are missing
- The transcription can only occur in the nucleus: fusion proteins must be
transported into the nucleus
=> Further analyses are required to confirm a newly discovered
protein-protein interaction
Can be extended to detect:
- protein-DNA interactions (yeast one-hybrid system)
- DNA-DNA interactions
Can be performed in other organisms: E. coli
Yeast two-hybrid system
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Predicting protein-protein interactions from databases
in silico
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Microscopy
fundamentals -> advanced
• Foundations of light microscopy / optical resolution
• Dark-field / phase contrast microscopy
• Fluorescence microscopy: advantages and limitations
• Confocal / multiphoton microscopy
• Total internal reflection microscopy
• Single molecule fluorescence microscopy
• Microscopy beyond the diffraction limit (STED / STORM)
• Fluorescence correlation spectroscopy
• Light sheet microscopy
Light microscopy
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What we can “see“
human
eye
light microscopy
scanning EM
transmission EM
scanning probe micrsocpy
atoms
organic
molecules
macromolecules eukaryotic
cells
viruses organismsbacteria
organelles
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Foundations of
light microscopy
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A very useful online guide to microsocpy:
https://www.microscopyu.com/microscopy-basics
Light microscopy: Upright microscope
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Light microscopy: Inverted microscope
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Condenser
Eyepiece
Observation tube
Stage
Objectives
Focus knob
Wave propagation:
Each point on a wavefront is the source of a new spherical wavelet.
The sum of these spherical wavelets forms the wavefront.
Valid for any type of wave: water waves, sound waves, electromagnetic waves (light).
Wellenberge
Huygens principle
wave crest
Light beam
perpendicular to
wave
area
50
Light is transmitted in various transparent materials
with different speeds (c):
Refractive index n = c0 / c1
Snellius law of refraction:
n1 sinα = n2 sinα‘
α: angle of incidence
α‘: emergent angle
=> relative to perpendicular
Light refraction
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Light refraction at air-glass interface
sinα
sinα'
=
n2
n1
Light entering an optically DENSER medium is refracted TOWARDS perpendicular.
Light entering an optically THINNER medium is refracted AWAY from perpendicular.
Light refraction
λ = wavelength
n2 = refractive
index of glass
n1 = refractive
index of air
α‘ = emgergent
angle
α = angle of
incidence
perpen-
dicular
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air
glass
Summary of wave propagation
53
When a light wave encounters an object, it may be reflected, absorbed, refracted,
diffracted, or scattered depending on the composition of the object and the
wavelength of light.
• Refraction: Light wave changes direction as it passes from one medium (n1) to another
medium (n2) as a result of differences in speed of light: in vacuum > air > water > glass.
• Reflection: Light wave hits an object and bounces off. Very smooth surfaces such as
mirrors reflect almost all incoming light.
• Diffraction: Interference or bending of waves around the corners of an obstacle or
through an aperture into the region of geometrical shadow of the obstacle/aperture.
=> All these phenomena can be explained by Huygens principle of wave propagation
Convex glass (spherical)
main plane
Brennpunkt
Optische Achse
Collective lens
optical axis
focal length
focal point
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focal plane
Concave glass (spherical)
Diverging lens
focal point
55
We obtain a real image (upside down) if object is placed:
(A) in focal plane (pobj = f): parallel rays emerge after lens; i.e. image is not focused
(B) between simple and double focal length (f < pobj < 2f): magnified image
(C) in double focal length (pobj = 2f): image has the same size as object
(D) beyond double focal length (pobj > 2f): demagnified image
Collective lens
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1
2
3
image
plane
optical axis
(1) parallel ray
(2) central ray
(3) focal ray
object
plane
(pobj)
f2x f f’
conjugated planes
Object placed between focal point and lens (pobj < f)
=> Diverging rays after lens, i.e. image cannot be focused
Collective lens
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Collective lens
Magnified virtual image behind object (loupe).
virtual
image
58
Object placed between focal point and lens (pobj < f)
=> Diverging rays after lens, i.e. image cannot be focused
Visual angel
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up to 200-fold magnification
Anton van Leeuwenhook (1632-1723)
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Combination of two collective lenses
Mtotal = Mobjective x Meye piece
Light path of combined microscope
virtual
image
object
objective
lens
eye piece
real
intermedate
image
61
Imaging light path of an optical microscope
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“upright“
microscope
intermediate
image
eye pieceOptical tube
length t
object
objective
back
focal plane
Setup of (historical) combined microscope
63
Setup of (modern) combined microscope
64
Modern microscopes are infinity corrected
65
finite optical
system
infinity-corrected
optical system
Conjugate planes in an optical microscope
66
=> Köhler
illumination
Bright-field microscopy
67
Light from the condenser passes through sample (transmission mode), is
attenuated by absorbing materials and collected by the objective
Total magnification (Mtot) = Mobjective x Meyepiece
• but there is a fundamental limit of resolution depending only on the objective:
λ/(2n*sinα) – note: M does not appear in this equation!
with λ: wavelength of light
n: refractive index
α: half of acceptance cone
• higher magnifications are called empty magnification
• The objective forms an image in the the intermediate image plane that
contains all information on the specimen accessible by the microscope! Any
further image magnification by eyepiece or camera lenses only changes the
size for easier observation or to fit the camera chip, but does not add any
information.
=> The resolution and brightness/contrast of an objective are essential
cell sample
examples:
- Gram-staining (bacteria)
- Stained tissues (histology)
but: fixing/staining kills cells
stained cells => higher contrast
Standard (bright field) microscopy
68
=> poor contrast because cells are
70% water
15% proteins
6% RNA
+ smaller amounts of others
Bright-field vs. Dark-field microscopy
69
objective
condenser
illumination
light
Condenser should
have larger NA
than the objective
Dark-field microscopy
70
Dark-field microscopy prevents non-diffracted light from entering the objective.
Only light rays diffracted by the specimen are collected by the objective. Thus,
a bright image appears against a dark background, resulting in a much better
image contrast compared to bright-field microscopy.
=> Enables observation of living cells/organisms.
In biology, dark-field microscopy has been replaced by improved techniques,
but it has recently reemerged for the analysis of strongly light scattering
(plasmonic) nanomaterials.
Amphipod crustacean (25x magnification)
Condenser
should have
larger NA
than objective