Download Radioisotopes in biology

Radioisotopes in biology
Particles produced by radioactive decay
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Types of radioactive decay
-α-particle emission (usually elements with high atomic number):
He2+ emitted
-positron emission (conversion of a proton to a neutron):
proton Æ neutron + β+
-neutron emission (conversion of a neutron to a proton):
neutron Æ proton + β-X-ray emission (fusion of proton and electron):
proton + electron Æ neutron + X-ray
-γ-ray emission does not change the atomic number or mass. It
usually accompanies the release of an α- or β- particle that leaves
the atom in an excited state. The atom can then reach a ground
state by releasing a γ-particle.
Radioactive decay energy
-Radioactive decay energy is measured in eV
-Definition: 1 eV is the energy absorbed by an electron in
accelerating through a potential difference of 1V.
α-particle (4-8 meV)
γ-rays and β-particle (usually less than 3 MeV)
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Half-lives of some isotopes used in
biological studies
Detection and quantification of radioactivity
-ionization of gases
-excitation of solids or solutions
-exposure of photographic emulsions
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Detection of radioactivity (by gas ionization)
1. A voltage is applied between the anode and cathode.
2. When a charged particle passing through the gas in the chamber
it ionizes particles.
3. A current is formed which is measured.
4. Can be used to measure α- and β- particles but not γ-rays.
Geiger-Müller tube
Townsend avalanche effect
Detection of radioactivity by excitation
-Liquid scintillation counting
-PM photomultiplier tube
Solvent e.g. toluene
Primary flour e.g. PPO= 2,5-diphenyl-oxazole
Secondary flour e.g. POPOP=1,4-bis(5-phenyloxazol-2-yl)benzene
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Working with radioactivity
Sheilding suitable for work with β-emitters
Geiger-müller counter
Scintillation proximity assay (SPA)
+
-High throughput screening
-Easy to automate
-Versatile method
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Exposure of photographic emulsions
-Autoradiography: To locate the position of a radiation source within a sample.
The sample is placed on a photographic emulsion and an image is produced
much as a normal photo.
Weak β-emittors are sutiable (e.g. 3H, 35S)
-Very sentitive method, exposure for days or longer
Tracing proteins in cells
EXAMPLE: a pulse chase experiment
1. PULSE Grow cells in medium containing radioisotope for a period of time (in this example we use
35S).
2. CHASE: Replace hot (radio-isotope containing)
medium with cold medium.
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Pulse chase
Example:
1. Take samples after certain times and isolate a desired biomolecule.
2. Quantify its amount of radioactivity
One type of antibody
fragment (diabody)
Another type of antibody
fragment (single chain)
Tracing proteins in multicellular
organisms
Example: Tracing tumor cells by SPECT imaging
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are needed to see this picture.
In Vivo Targeting–Experimental Set-up–Professor Stahl.
SK-OV-3 s.c. xenograft tumors
SPECT (Single Photon Emission Computed Tomography)
125I
QuickTime™ and a
GIF decompressor
are needed to see this picture.
HER2-targeting - effect of affinity
ZHER2:4 - 50 nM
6 hours
a
ZHER2:342 - 22 pM
6 hours
b
ZHER2:342 - 22 pM
24 hours
c
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GIF decompressor
are needed to see this picture.
Biodistribution in mice
Biodistribution (125I, n=4, 12h p.i.)
brain
bone
muscle
skin
tumor
thyroid/organ
saliv gland
larg int
sm int
stomach
pancreas
spleen
liver
lung
heart
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kidney
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blood
%ID/g
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Protein-ligand interactions
-interactions are influenced by physical
parameters such as pH, temperature and ionic
concentration.
-It is important to allow the system to reach
equilibrium
The dissociation constant Kd for a particular
interaction can be determined experimentally,
through e.g. a scatchard plot.
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Equlibrium dialysis
Surface plasmon resonance (SPR)
spectroscopy
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Coupling to of ligands to the
sensorchip
Association and dissociation
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Quartz crystal microbalance (QCM)
biosensor
Resonance frequency is changing as a result of mass deposition on the surface
Electrophoretic mobility shift assay
(EMSA)
To analyze the interaction between protein and DNA/RNA
1. Radio-label the DNA (typically using 32P), but e.g.
fluorescent probe is also ok.
2. Mix a fixed amount of DNA with a varying amount of protein
3. Separate the Protein DNA mixtures by SDS-PAGE.
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EMSA
DNA/Protein
complex
DNA
Kd can be directly determined when half the DNA molecules
are bound to Protein
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