Radioisotopes in biology. Particles produced by radioactive decay
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1 Radioisotopes in biology Particles produced by radioactive decay 1
2 Types of radioactive decay -α-particle emission (usually elements with high atomic number): He 2+ emitted -positron emission (conversion of a proton to a neutron): proton neutron + β + -neutron emission (conversion of a neutron to a proton): neutron proton + β - -X-ray emission (fusion of proton and electron): proton + electron 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) 2
3 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 3
4 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 4
5 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 5
6 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. 3 H, 35 S) -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. 6
7 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 7
8 QuickTime and a GIF decompressor 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) 125 I QuickTime and a GIF decompressor are needed to see this picture. HER2-targeting - effect of affinity ZHER2:4-50 nm ZHER2: pm ZHER2: pm 6 hours 6 hours 24 hours a b c 8
9 QuickTime and a GIF decompressor are needed to see this picture. 10 Biodistribution in mice Biodistribution ( 125 I, n=4, 12h p.i.) 5 0 blood heart lung liver spleen pancreas kidney stomach sm int larg int saliv gland thyroid/organ tumor skin muscle bone brain %ID/g 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 K d for a particular interaction can be determined experimentally, through e.g. a scatchard plot. 9
10 Equlibrium dialysis Surface plasmon resonance (SPR) spectroscopy 10
11 Coupling to of ligands to the sensorchip Association and dissociation 11
12 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 32 P), 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. 12
13 DNA/Protein complex EMSA DNA Kd can be directly determined when half the DNA molecules are bound to Protein 13
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