Protection Assay

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A Protection Assay (in German protection test ) refers to a biochemical method for the determination of protected and thus for enzymes , chemicals or markings inaccessible molecular structures .

principle

In the protection assay , biomolecules are subjected to a treatment, for example incubation with an enzyme. The protection assays are based on the limited accessibility of covered molecular areas. The molecule to be examined can thus be subjected to different conditions (e.g. inhibitors , denaturing temperatures, salts , crosslinkers ). The comparison with the negative control allows conclusions to be drawn about a corresponding change. The biomolecule can also be incubated to detect an interaction with another molecule. In this case, the changed accessibility allows indications of a binding and possibly also of the binding site .

Proteins

For proteins z. B. in a limited proteolysis assay (synonym protease exclusion assay ) different proteases are used to break down accessible protein structures . Due to the protein folding of the protein to be broken down, some proteases can only break down areas close to the surface. In particular, proteases with a less exposed (ie lower lying) active center can only degrade unfolded areas of their substrate for spatial reasons. With a change in temperature which may thermostability of a protein are examined, as the temperature increases, the protein is unfolded. This is a variant of the Thermal Shift Assay . The limited proteolysis also allows important findings after ligand binding. The changed accessibility after the incubation of nuclear receptors with the specific hormone or with a DNA target sequence allows indications of allosteric changes.

DNA

Nucleic acids are z. B. subjected to a nuclease protection assay to examine nucleosomes or a DNAse footprinting assay to examine specific protein-DNA interactions . So are z. B. DNA strands wound around histones or DNA sequences covered by DNA-binding proteins are not accessible to some nucleases , while unbound DNA segments can be degraded. This shows z. B. with nucleosomes in an agarose gel electrophoresis after partial degradation of the DNA a DNA ladder . The binding sequences can also be identified by incubating protein-DNA complexes with exonucleases .

RNA

In an RNase protection assay , RNAs are protected from degradation by single-strand degrading RNases with specifically binding RNA hybridization probes . This enables a transcription start point to be identified. An RNase protection assay can be used to protect a molecule during an RNA purification with digestion of all further RNA molecules of a different sequence. The absence of other RNA sequences reduces the detection limit in an RT-PCR because the specificity of the primer binding is increased.

DNA / RNA

A classic method in molecular biology is the S1 assay . In this method for the investigation of DNA-RNA hybrids, the single-strand-specific nuclease S1 from Aspergillus oryzae is used . In a DNA-RNA hybrid, S1 cuts the unhybridized single-stranded sections at the ends of the hybrid. The result is a fully double-stranded hybrid. The investigation uses radioactively labeled DNA at the 5 'end, e.g. B. comprises the start of transcription of a gene. For size analysis, the protected DNA is separated with a size marker or the sequence reaction of the DNA sample on a denaturing polyacrylamide gel .

Individual evidence

  1. ↑ SR Nirantar, X. Li, JW Siau, FJ Ghadessy: Rapid screening of protein-protein interaction inhibitors using the protease exclusion assay. In: Biosensors & Bioelectronics . [Electronic publication before going to press] January 2014, ISSN  1873-4235 . doi : 10.1016 / j.bios.2013.12.060 . PMID 24508816 .
  2. ↑ RS Jamison, ME Newcomer, DE Ong: Detection of conformational changes in cellular retinoid-binding proteins by limited proteolysis. In: Methods in molecular biology (Clifton, NJ). Volume 89, 1998, pp. 165-176, ISSN  1064-3745 . doi : 10.1385 / 0-89603-438-0: 165 . PMID 9664327 .
  3. ↑ A. Fontana, PP de Laureto, B. Spolaore, E. Frare, P. Picotti, M. Zambonin: Probing protein structure by limited proteolysis. In: Acta biochimica Polonica. Volume 51, Number 2, 2004, pp. 299-321, ISSN  0001-527X . PMID 15218531 . PDF .
  4. ↑ S. Suzuki, S. Nishida, K. Ohno, T. Santa: Modulation of coactivator recruitment by cooperative ligand binding to human estrogen receptor alpha and beta. In: Biological & pharmaceutical bulletin. Volume 30, Number 9, September 2007, pp. 1641-1647, PMID 17827713 .
  5. ↑ J. Brodie, IJ McEwan: Intra-domain communication between the N-terminal and DNA-binding domains of the androgen receptor: modulation of androgen response element DNA binding. In: Journal of molecular endocrinology. Volume 34, Number 3, June 2005, pp. 603-615, doi : 10.1677 / jme.1.01723 , PMID 15956332 .
  6. ^ A. Vora, DP Grandgenett: DNase protection analysis of retrovirus integrase at the viral DNA ends for full-site integration in vitro. In: Journal of virology. Volume 75, Number 8, April 2001, pp. 3556-3567, ISSN  0022-538X . doi : 10.1128 / JVI.75.8.3556-3567.2001 . PMID 11264345 . PMC 114847 (free full text).
  7. ↑ M. Mizuuchi, TA Baker, K. Mizuuchi: DNase protection analysis of the stable synaptic complexes involved in Mu transposition. In: Proceedings of the National Academy of Sciences . Volume 88, Number 20, October 1991, pp. 9031-9035, ISSN  0027-8424 . PMID 1656459 . PMC 52645 (free full text).
  8. ↑ a b M. F. Carey, CL Peterson, ST Smale: The RNase protection assay. In: Cold Spring Harbor protocols. Volume 2013, number 3, March 2013, p., ISSN  1559-6095 . doi : 10.1101 / pdb.prot071910 . PMID 23457339 .
  9. ↑ A. Sandelin, P. Carninci, B. Lenhard, J. Ponjavic, Y. Hayashizaki, Hume DA: Mammalian RNA polymerase II promoter core: insights from genome-wide studies. In: Nature Reviews Genetics . Volume 8, Number 6, June 2007, pp. 424-436, ISSN  1471-0056 . doi : 10.1038 / nrg2026 . PMID 17486122 .
  10. ↑ AD Paradh, AE Hill, WJ Mitchell: Detection of beer spoilage bacteria Pectinatus and Megasphaera with acridinium ester labeled DNA probes using a hybridisation protection assay. In: Journal of microbiological methods. Volume 96, January 2014, pp. 25-34, ISSN  1872-8359 . doi : 10.1016 / j.mimet.2013.10.014 . PMID 24184310 .
  11. ↑ HA Young, JJ Subleski, SM Krebs: Multiprobe ribonuclease protection assay for simultaneous measurement of mRNA expression. In: Current protocols in immunology / edited by John E. Coligan ... [et al.]. Chapter 10 August 2003, S. Unit 10.29, ISSN  1934-368X . doi : 10.1002 / 0471142735.im1029s54 . PMID 18432896 .
  12. ↑ F. Gannon, JM Jeltsch, F. Perrin: A detailed comparison of the 5'-end of the ovalbumin gene cloned from chicken oviduct and erythrocyte DNA. In: Nucleic acids research. Volume 8, Number 19, October 1980, pp. 4405-4421, PMID 6253917 , PMC 324248 (free full text).
  13. ^ RC Wiegand, GN Godson, CM Radding: Specificity of the S1 nuclease from Aspergillus oryzae. In: The Journal of biological chemistry. Volume 250, Number 22, November 1975, pp. 8848-8855, PMID 171268 .