Heat stabilization

from Wikipedia, the free encyclopedia

Heat stabilization describes an effect in which heat leads to an increased conservation of biological tissues . It is used in biochemistry for the short-term preservation of biomolecules.

properties

In the case of heat stabilization, the tissue to be prepared is heated in a buffer solution by conduction to over 80 ° C, usually to 95 ° C. By heating among other things, it contains are proteins denatured , including autolysis causing enzymes such as peptidases , nucleases , glycosidases and lipases . At the temperatures used, this also results in partial pasteurization . The temperature used is below the boiling point of the buffer used in order to avoid the formation of bubbles within the cells , which can break up the cell structures . Broken cells make it difficult to recognize structures during a possible microscopic examination, and some cytosolic proteins would prematurely diffuse out of the cells into the buffer before a specific extraction is carried out.

In contrast to chemical fixation with fixatives, heat stabilization is a physical process, which is why no antigen unmasking is required before immunostaining . However, due to the denaturation of the proteins during immunostaining, primarily continuous epitopes are recognized by antibodies, while discontinuous epitopes are partially refolded by denaturation , then only fold back incompletely and are therefore often not recognized by specific antibodies. Heat stabilization is only suitable for long-term storage of biological samples to a limited extent , as some proteins can still be broken down afterwards . Compared to the shock freezing of samples in liquid nitrogen, the heat stabilization preserves some phosphoproteins better and others worse, which is why the method to be used is determined experimentally in each individual case.

Applications

The advantage of heat stabilization compared to preservation by fixation lies in the fact that the molecules contained in the sample do not change due to cross-linking , which means that antigen unmasking before further analysis is not required. The proteins in heat-stabilized samples can be further characterized by mass spectrometry (especially phosphoproteins), Western blotting , 1D and 2D gel electrophoresis and reversed-phase protein microarrays . An extraction of the proteins is usually a chaotrope performed in the extraction buffer and is especially suitable for phosphoproteins.

history

The heat stabilization of biological tissues for research purposes was first used in 1994 by MZ Hossain, using microwaves . Previously, thermal denaturation was used in food production for the heat stabilization of raw food, for the preservation of wine and in biochemical analysis for the denaturation and inactivation of individual proteins, e.g. B. for sample preparation in SDS-PAGE or to terminate a restriction digestion or reverse transcription .

Individual evidence

  1. ↑ K. Kultima, K. Sköld, M. Borén: Biomarkers of disease and post-mortem changes - Heat stabilization, a necessary tool for measurement of protein regulation. In: Journal of proteomics. Volume 75, Number 1, December 2011, pp. 145-159, ISSN  1876-7737 . doi : 10.1016 / j.jprot.2011.06.009 . PMID 21708298 .
  2. ↑ M. Svensson, M. Boren, K. Sköld, M. Fälth, B. Sjögren, M. Andersson, P. Svenningsson, PE Andren: Heat stabilization of the tissue proteome: a new technology for improved proteomics. In: Journal of proteome research. Volume 8, Number 2, February 2009, pp. 974-981, ISSN  1535-3893 . doi : 10.1021 / pr8006446 . PMID 19159280 .
  3. ↑ T. Gemoll, O. Löwe, M. Borén, M. Oberländer, S. Hartwig, S. Lehr, UJ Roblick, G. Auer, H. Jörnvall, JK Habermann: The impact of pre-analytical conditions on the serum proteome : heat stabilization versus nitrogen storage. In: Archives of physiology and biochemistry. Volume 119, Number 3, July 2013, pp. 100-107, ISSN  1744-4160 . doi : 10.3109 / 13813455.2013.806556 . PMID 23826811 .
  4. ↑ Md. Mahiuddin Ahmeda, Katheleen J. Gardiner: Preserving protein profiles in tissue samples: differing outcomes with and without heat stabilization. In: Journal of neuroscience methods. Volume 196, Number 1, March 2011, pp. 99-106, ISSN  1872-678X . doi : 10.1016 / j.jneumeth.2011.01.004 . PMID 21236297 . PMC 3299412 (free full text).
  5. ↑ Alicia Lundby, Anna Secher, Kasper Lage, Nikolai B. Nordsborg, Anatoliy Dmytriyev, Carsten Lundby, and Jesper V. Olsen: Quantitative maps of protein phosphorylation sites across 14 different rat organs and tissues. In: Nature Communications . Volume 3, 2012, p. 876, ISSN  2041-1723 . doi : 10.1038 / ncomms1871 . PMID 22673903 . PMC 3621391 (free full text).
  6. ^ GB Smejkal, C. Rivas-Morello, JH Chang, E. Freeman, AJ Trachtenberg, A. Lazarev, AR Ivanov, WP Kuo: Thermal stabilization of tissues and the preservation of protein phosphorylation states for two-dimensional gel electrophoresis. In: Electrophoresis. Volume 32, Number 16, August 2011, pp. 2206-2215, ISSN  1522-2683 . doi : 10.1002 / elps.201100170 . PMID 21792998 .
  7. ↑ C. Spellman, MM Ahmed, D. Dubach, KJ Gardiner: Expression of trisomic proteins in Down syndrome model systems. In: Genes. Volume 512, Number 2, January 2013, pp. 219-225, ISSN  1879-0038 . doi : 10.1016 / j.gene.2012.10.051 . PMID 23103828 .
  8. ↑ Md. Mahiuddin Ahmed, Ranjitha Dhanasekaran, Suhong Tong, Frances K. Wiseman, Elizabeth MC Fisher, Victor LJ Tybulewicz, Katheleen J. Gardiner: Protein profiles in Tc1 mice implicate novel pathway perturbations in the Down syndrome brain. In: Human Molecular Genetics . Volume 22, Number 9, May 2013, pp. 1709-1724, ISSN  1460-2083 . doi : 10.1093 / hmg / ddt017 . PMID 23349361 . PMC 3613160 (free full text).
  9. ↑ OA Kofanova, F. Fack, SP Niclou, F. Betsou: Combined effect of tissue stabilization and protein extraction methods on phosphoprotein analysis. In: Biopreservation and biobanking. Volume 11, Number 3, June 2013, pp. 161-165, ISSN  1947-5543 . doi : 10.1089 / bio.2013.0008 . PMID 24850093 .
  10. ↑ MZ Hossain, LJ Murphy, EL Hertzberg, JI Nagy: Phosphorylated forms of connexin43 predominate in rat brain: demonstration by rapid inactivation of brain metabolism. In: Journal of neurochemistry. Volume 62, Number 6, June 1994, pp. 2394-2403, ISSN  0022-3042 . PMID 8189244 .
  11. ↑ George FM Ball: Vitamins In Foods: Analysis, Bioavailability, and Stability. CRC Press 2005. ISBN 9781420026979 .