Autoradiography

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Autoradiogram of a section through the brain of a rat embryo. The labeling was carried out with oligonucleotide sequences which were conjugated with 35 S -dATP ( deoxyadenosine triphosphate ) and which bind to GAD67 ( glutamate decarboxylase 67). The areas with high radioactivity (high marker concentration) are black. This is particularly the case in the subventricular zone (SVZ). The black scale bar corresponds to a length of 2 mm.

Autoradiography or radiography (common abbreviation AURA) describes the visualization of a chemical component by radioactive nuclides , originally by blackening a photographic film, now increasingly with the help of a radiation detector . The image obtained is called an autoradiogram .

Applications

The autoradiography was over three decades, an integral part of DNA sequencing by Frederick Sanger . One of the protagonists was the pathologist Elmar Stöcker . 48 of his publications are based on autoradiography. Since the last years of the 20th century, however, fluorescent instead of radioactively labeled DNA nucleotides have increasingly been used for sequence analysis. Autoradiography is also used in the production of recombinant proteins, analysis of enzymatic reactions, or identification of enzyme substrates. It is also used in pharmacokinetics to e.g. B. Liberation Absorption Distribution Metabolism Excretion Studies (LADME). The combination of autoradiography and neutron activation is used as neutron autoradiography to investigate local elemental compositions in paintings.

execution

For autoradiography, radioactive nuclides must be introduced into the molecules to be analyzed. Due to the frequent occurrence of the corresponding stable isotopes in biomolecules and their relatively low level of danger, the radionuclides 14 C ( carbon ), 35 S ( sulfur ), 32 P ( phosphorus ) and 3 H ( tritium ) are often used.

Example 1 - labeling of recombinant proteins

A bacterial strain transformed with an expression plasmid is treated with a nutrient medium which contains the radioactively labeled amino acid 35 S- methionine . 35 S-methionine is incorporated into the recombinant protein encoded on the plasmid . A bacterial extract is separated by SDS-PAGE , the gel is dried and a film is applied. After the film has been “exposed” to the beta rays emitted by the sulfur-35, the position of the recombinant protein is visible on the developed film.

2nd example - analysis of enzyme activity

An ATPase - an enzyme that splits ATP - is incubated with radioactively labeled 32 P-ATP. The mixture is separated after different times by thin layer chromatography , the chromatography plate is dried and placed on film. The blackening of the film by 32 P- phosphate reflects the activity of the enzyme.

literature

  • A. Scholl, E. Stöcker, H. Finger and D. Krammenschneider: Morphological investigation of the mouse spleen after cyclophosphamide application . Morphometric methods compared with autoradiographic methods . Rat. Dtsch. Ges. Path. 61 (1977), p. 377.
  • P.-H. Bippus, E. Stöcker and H. Heyn: Cellular 3 H-TdR activity measurement and topicality of regenerating cells in kidney and liver after successive uninephrectomy and two-thirds part hepatectomy (autoradiography). Rat. Dtsch. Ges. Path. 61 (1977), p. 436.

Individual evidence

  1. ^ EG Solon, A. Schweitzer, M. Stoeckli, B. Prideaux: Autoradiography, MALDI-MS, and SIMS-MS imaging in pharmaceutical discovery and development. In: The AAPS Journal . Volume 12, Number 1, March 2010, pp. 11-26, ISSN  1550-7416 . doi : 10.1208 / s12248-009-9158-4 . PMID 19921438 . PMC 2811645 (free full text).
  2. ↑ R. Wester Meier, R. Marouga: protein detection methods in proteomics research. In: Bioscience Reports . Volume 25, Numbers 1-2, 2005 Feb-Apr, pp 19-32, ISSN  0144-8463 . doi : 10.1007 / s10540-005-2845-1 . PMID 16222417 .
  3. ↑ DP Holschneider, JM Maarek: Brain maps on the go: functional imaging during motor challenge in animals. In: Methods (San Diego, Calif.). Volume 45, Number 4, August 2008, pp. 255-261, ISSN  1095-9130 . doi : 10.1016 / j.ymeth.2008.04.006 . PMID 18554522 . PMC 2561174 (free full text).