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By Martina Newell / Burke, James I McGloughlin

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1–1 % TFA/FA (TFA is frequently used for the MALDI approach, whereas FA is used for ESI technique). 1% TFA/FA. The pooled fractions are partially dried to concentrate the sample and remove the MeCN if a combined approach with LC-MS is used. Bonetto et al. (Bonetto, Bergman, Jörnwall, & Sillard, 1997) proposed alkylating peptides after the tryptic digestion using 2-bromoethyl-trimethylammonium (Itano & Robinson, 1972). Such modification of the cysteine-containing peptides facilitates carboxypeptidase P and Y degradation.

V. BIENVENUT techniques are limited to method development much more than protein quantitation, which is more accurate using 35S AAs. 3. Denaturing staining process Some extremely sensitive staining methods are available for visualising gelseparated polypeptides on a membrane. Unfortunately, it is generally impossible to reuse such membranes for further analysis such as immuno-detection. , 1995). The intermediate solution is to produce a duplicate of the membrane (from a single gel (Neumann & Mullner, 1998) or from two parallel-prepared gels).

Another common protein treatment before endoproteolytic digestion is reduction followed by alkylation of the sulfhydryl groups. After this modification, the primary sequence of the proteins is easily available to the digesting enzyme. g. , 1999). Sulfhydryl groups can also react during the gel separation process with the non-polymerised acrylamide (Bordini, Hamdan, & Righetti, 2000). The utilisation of polymeric membranes shows the great advantage of maintaining the protein substrate at the surface of the membrane and the excess of reagent can easily washed.

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