Gel extraction

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The gel extraction includes biochemical methods for the isolation of biomolecules such as DNA , RNA and proteins from agarose gels or polyacrylamide gels .

principle

Agarose gel with ethidium bromide on a UV transilluminator when cutting out

The piece of gel with the colored band to be examined is cut out of the gel with a scalpel or punch and treated with one of the following methods. The methods can be subdivided according to their respective separation principle. In the case of using fluorescent dyes, irradiation with UV light , e.g. B. on a transilluminator (UV light table), kept as short as necessary, since the irradiation can lead, among other things, to the formation of thymine dimers or strand breaks. The nucleic acids damaged by UV light can partially inhibit subsequent reactions. When using non -fluorescent dyes such as methylene blue or crystal violet , the cutting out takes place without a UV transilluminator under daylight. When using a crystal-violet-colored gel, the sample buffer should not contain bromophenol blue or xylene cyanol blue, as the electrophoretic separation suffers from the interaction of these dyes.

extraction

Agarose gels can be dissolved by adding a buffer with chaotropes and heating to about 55 ° C. The chaotropic salts denature the proteins and dissolve the agarose gel matrix and keep it in solution. Sodium iodide is mostly used as a chaotrope . In the preceding agarose gel electrophoresis and in the gel, no TBE buffer should be used for a chemical gel extraction, as the borate it contains reduces the chaotropic effect of the sodium iodide. The dissolved gel is then used in a DNA extraction process to isolate the DNA it contains.

In mass spectrometry , after an in-gel digestion, the cleaved protein fragments are extracted from polyacrylamide gels , e.g. B. with an ammonium hydrogen carbonate buffer or with a solution of formic acid , acetonitrile and sometimes isopropanol .

Freeze

The gel piece is frozen at −20 ° C. The piece of gel is then folded into a piece of plastic film (usually Parafilm ). By squeezing the frozen gel piece in the foil, the gel is crushed by the ice crystals it contains. The liquid released from this with the molecule to be examined is then transferred to a new reaction vessel. This is why the method is also known as Freeze and Squeeze (English for 'freezing and squeezing'). To avoid unwanted entrainment of gel residues, the solution is centrifuged for up to 30 seconds, if necessary, and the supernatant solution is transferred to a new reaction vessel. The solution is then used for ethanol precipitation in order to isolate the DNA it contains.

Electroelution

The electroelution takes place in a gel elution electrophoresis chamber while a voltage is applied. Just as charged molecules migrate into a gel during gel electrophoresis, they can also migrate out of the gel into a solution .

literature

Individual evidence

  1. ^ NK Rand: Crystal Violet Can Be Used to Visualize DNA Bands During Gel Electrophoresis and to Improve Cloning Efficiency . In: Elsevier Trends Journals Technical Tips Online (1996), T40022.
  2. ↑ TA Borodina, H. Lehrach , AV Soldatov: DNA purification on homemade silica spin columns. In: Anal Biochem. , Vol. 321, No. 1, 2003, pp. 135-7, PMID 12963065 .
  3. ↑ B. Vogelstein, D. Gillespie: Preparative and analytical purification of DNA from agarose. In: Proc Natl Acad Sci US A. Vol. 76, No. 2, 1979, pp. 615-619, PMID 284385 , PMC 382999 (free full text).
  4. ↑ A. Shevchenko, M. Wilm, O. Vorm, M. Mann: Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. In: Analytical chemistry. Volume 68, Number 5, March 1996, ISSN  0003-2700 , pp. 850-858, PMID 8779443 .
  5. ↑ F. Gharahdaghi, CR Weinberg, DA Meagher, BS Imai, SM Mische: Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: a method for the removal of silver ions to enhance sensitivity. In: Electrophoresis. Volume 20, number 3, March 1999, ISSN  0173-0835 , pp. 601-605, doi : 10.1002 / (SICI) 1522-2683 (19990301) 20: 3 <601 :: AID-ELPS601> 3.0.CO; 2- 6 , PMID 10217175 .
  6. ^ J Havlis, H. Thomas, M. Sebela, A. Shevchenko: Fast-response proteomics by accelerated in-gel digestion of proteins. In: Analytical chemistry. Volume 75, Number 6, March 2003, ISSN  0003-2700 , pp. 1300-1306, PMID 12659189 .
  7. ↑ CR Jiménez, L. Huang, Y. Qiu, AL Burlingame: In-gel digestion of proteins for MALDI-MS fingerprint mapping. In: Current protocols in protein science / editorial board, John E. Coligan ... [et al.]. Chapter 16, May 2001, S. Unit 16.4, ISSN  1934-3663 , doi : 10.1002 / 0471140864.ps1604s14 , PMID 18429131 .
  8. ↑ A. Shevchenko, H. Tomas, J. Havlis, JV Olsen, M. Mann: In-gel digestion for mass spectrometric characterization of proteins and proteomes. In: Nature protocols. Volume 1, Number 6, 2006, ISSN  1750-2799 , pp. 2856-2860, doi : 10.1038 / nprot.2006.468 , PMID 17406544 .
  9. ↑ H. Ehring, S. Strömberg, A. Tjernberg, B. Norén: Matrix-assisted laser desorption / ionization mass spectrometry of proteins extracted directly from sodium dodecyl sulphate-polyacrylamide gels. In: Rapid communications in mass spectrometry: RCM. Volume 11, number 17, 1997, ISSN  0951-4198 , pp. 1867-1873, doi : 10.1002 / (SICI) 1097-0231 (199711) 11:17 <1867 :: AID-RCM46> 3.0.CO; 2-Z , PMID 9404036 .
  10. ↑ RW Thuring, JP Sanders, P. Borst: A freeze-squeeze method for recovering long DNA from agarose gels. In: Analytical biochemistry. Volume 66, Number 1, May 1975, ISSN  0003-2697 , pp. 213-220, PMID 1096670 .
  11. ↑ D. Tautz, M. Renz: An optimized freeze-squeeze method for the recovery of DNA fragments from agarose gels. In: Analytical biochemistry. Volume 132, Number 1, July 1983, ISSN  0003-2697 , pp. 14-19, PMID 6312834 .
  12. ↑ H. Seelert, F. Krause: Preparative isolation of protein complexes and other bioparticles by elution from polyacrylamide gels. In: Electrophoresis. Volume 29, Number 12, June 2008, ISSN  0173-0835 , pp. 2617-2636, doi : 10.1002 / elps.200800061 , PMID 18494038 .
  13. ↑ D. Moore, J. Chory, RK Ribaudo: Isolation and purification of large DNA restriction fragments from agarose gels. In: Current protocols in immunology / edited by John E. Coligan ... [et al.]. Chapter 10 May 2001, S. Unit 10.5, ISSN  1934-368X , doi : 10.1002 / 0471142735.im1005s08 , PMID 18432696 .