Radiation Uses in Biomedical Research

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Radiation Uses in Biomedical Research
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Types of Radiation Uses

• X-Rays
• There obvious use- medical imaging
• Study of molecular structure X-ray diffraction
• Study of molecular composition X-ray fluorescence

• Isotopes
• In vivo labeling and tracer studies
• In vitro labeling and localization studies
• Membrane transport / uptake studies

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X-ray diffraction
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Protein Structural Analysis
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A famous X-ray crystallograph

• Safety issues?
• Usually these are lower energy X-rays
• Normally the instruments have lead-plastic
  shielding
• Usually put a badge in the room for area
  monitoring.

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Radioactive Materials in biomedical research

• Most commonly used radioisotopes
• H3, C14, P32, P33, S35 -- all beta emitters
• In vivo and in vitro labeling
• Na22, K40, Ca45, I125 -- all beta/gamma emitters
• Uptake studies for the ions, in vitro labeling
  for I125

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Typical amounts of radioactive nuclides used as
tracers in biological experiments

• H3 10 µCi (22000 dpm) 5 mCi (107 dpm)
• C14 5 µCi 1 mCi
• P32 50 µCi to 200 µCi
• S35 50 µCi to 1 mCi

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Some uses for tracers in experiments

• Labeling/detection of small molecules
• Study metabolic pathways
• Study uptake/transport of molecules
• Study reaction mechanisms
• Labeling/detection of macromolecules
• Study synthesis of populations of molecules
• Detect specific macromolecules
• radioactive antibodies
• radiolabeled DNA probes

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An example of an uptake study Weise et al.2000.
Plant Cell 121345
Figure 3. Kinetic Analysis of 14C-Sucrose Uptake
by Yeast Strain SUSY7/ura3 Expressing AtSUT4.
(A) Time course of sucrose uptake. SUSY7/ura3
yeast transformed with StSUT4 in pDR195, AtSUT4
in pDR196, or empty pDR195/pDR196 vector were
assayed for 14C-sucrose uptake at 1 mM sucrose
and pH 4.0. (B) Rate of sucrose uptake by
SUSY7/ura3 expressing AtSUT4. Transport assays
were performed at pH 5.5, and uptake rates are
plotted against final sucrose concentrations in
the assays. Background uptake rates (empty
vector) were subtracted.
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Question How did they know how much radioactive
sucrose was in the cells?
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Liquid Scintillation Counters
3H
14C
32P
log Scale
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In vivo Labeling of Proteins

• Use H3-labeled Leu
• Cheap
• Low energy
• Low specific activity
• Use C14-labeled Leu
• More expensive
• More energy
• Low specific activity
• Use S35-labeled Met
• Similar to C14
• More energy still
• Higher specific activities

• Can find H3 and C14 only by LSC---cant identify
  individual proteins
• S35 detectable using film-based imaging can see
  individual labeled bands in an electrophoresis
  gel.

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Gel electrophoresis of proteins
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Autoradiography
Beta particle
Radiolabeled protein
Gel
X-ray film
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In vivo labeled protein experiment
Secretion of Hsp150 in sec13-1 mutant. (A)
Sec13-1 cells (H230) were preincubated for 15 min
and pulse labeled for 5 min at 37C with
35Smethionine/cysteine (lanes 1 and 2).
Parallel cell samples were chased in the presence
of CHX at 37C. Medium (m) and cell lysate (c)
samples were immunoprecipitated with Hsp150
antiserum followed by SDS-PAGE analysis. ER (100
kDa) and mature forms (150 kDa) of Hsp150 are
indicated.
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Labeling DNA

• Internal labels
• a-P32 dNTP
• Nick translation
• Random Primer
• End Label
• ?-P32 dNTP
• Terminal transferase

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Fixed contamination surveys

• Hold probe about 1-2 cm from surface
• Move horizontally at about 1-2 cm/sec
• Pinpoint, mark, and decontaminate any activity
  above the limits
• Regs gt5000 dpm ß/100 sq. cm.
• ALARA gt100 dpm ß/100 sq. cm.