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The in-gel digestion is part of the sample preparation for the mass spectrometric identification of proteins in course of proteomic analysis. The method was introduced 1992 by Rosenfeld.〔Rosenfeld, J et al., ''Anal Biochem'', 1992, 203 (1), 173-9.〕 Innumerable modifications and improvements in the basic elements of the procedure remain.〔Hellman, U et al., ''Anal Biochem'', 1995, 224 (1), 451-455.〕〔Jeno, P et al., ''Anal Biochem'', 1995, 224 (1), 75-82.〕〔Shevchenko, A et al., ''Anal Chem'', 1996, 68 (5), 850-8.〕〔Borchers, C et al., ''Anal Chem'', 2000, 72 (6), 1163-8.〕〔Shevchenko, A et al., ''Nat Protoc'', 2006, 1 (6), 2856-60.〕〔(Granvogl, B et al., ''Proteomics'', 2007, 7 (5), 642-54. )〕 The in-gel digestion primarily comprises the four steps destaining, reduction and alkylation (R&A) of the cysteines in the protein, proteolytic cleavage of the protein and extraction of the generated peptides. ==Destaining== Proteins which were separated by 1D or 2D PAGE are usually visualised by staining with dyes like Coomassie Brilliant Blue (CBB) or silver. Although the sensitivity of the method is significantly lower, the use of Coomassie is more common for samples destined for mass spectrometry since the silver staining impairs the analysis. After excision of the protein band of interest from the gel most protocols require a destaining of the proteins before proceeding. The destaining solution for CBB contains usually the buffer salt ammonium bicarbonate (NH4HCO3) and a fraction of 30%-50% organic solvent (mostly acetonitrile). The hydrophobic interactions between protein and CBB are reduced by the organic fraction of the solution.〔Jin, Y and Manabe, T, ''Electrophoresis'', 2005, 26 (6), 1019-28.〕 At the same time, the ionic part of the solution diminishes the electrostatic bonds between the dye and the positively charged amino acids of the protein. In contrast to a mixture of water with organic solvent the effectivity of destaining is increased. An increase of temperature promotes the destaining process.〔Lloyd, MD, ''Anal Biochem'', 1996, 241 (1), 139-40.〕 To a certain degree (< 10%) the destaining procedure is accompanied with a loss of protein.〔Speicher, KD et al., ''Journal of Biomolecular Techniques'', 2000, 11 (2), 74-86.〕 Furthermore, the removal of CBB does not affect the yield of peptides in the mass spectrometric measurement.〔〔Terry, DE et al., ''J Am Soc Mass Spectrom'', 2004, 15 (6), 784-94.〕 In the case of silver stained protein bands the destaining is accomplished by oxidation of the metallic silver attached to the protein by potassium ferricyanide or hydrogen peroxide (H2O2).〔Gharahdaghi, F et al., ''Electrophoresis'', 1999, 20 (3), 601-5.〕〔Sumner, LW et al., ''Rapid Commun Mass Spectrom'', 2002, 16 (3), 160-8.〕 The released silver ions are complexed subsequently by sodium thiosulfate. 抄文引用元・出典: フリー百科事典『 ウィキペディア（Wikipedia）』 ■ウィキペディアで「In-gel digestion」の詳細全文を読む スポンサード リンク
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