By Professor Saul B. Needleman (auth.)
Confusion now hath made his masterpiece Macbeth II iii seventy two Whence and what are these execrable form? Paradise misplaced Ib 1 681 Confusion worse confounded Paradise misplaced Ib 1 995 while the manuscript for the 1st a part of this ebook used to be proposed, it was once expected that the dialogue of the total box of protein se quencing will be lined in one quantity - from purification and characterization of the protein via fragmentation through chemical or enzymic skill and, eventually, to reassembly of the pointed out person peptides into the reconstructed overall series. It quickly grew to become obtrusive that this might no longer be attainable. whereas the motive was once to limit the reveal of techniques basically to that details concerned about "hands on" rainy chemistry, it turned obvious thorough presentation will require, additionally, a dialogue of convinced instrumental and extra theoretical ways now not integrated within the first quantity. additionally, the whole knowing of the sphere of protein sequencing has complicated significantly because the inception of this e-book. the aim of the 1st quantity was once to supply functional details in adequate aspect to allow the researcher to adopt the particular sequencing approaches in his personal laboratory.
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Polystyrene-based resins suffer from the problem that they swell and shrink in different solvents, which can lead to channeling or blocking when the resin beads are packed into reaction columns. This difficulty can be avoided by mixing the resin with a large (20-fold) excess of glass beads, so that swelling and shrinking occurs in the interstices between the glass beads (249). Alternatively, reactions can be carried out in a reaction vessel, rather than a column, though this leads to some mechanical problems (140).
COOH I' ~C-NH-CH-C-NH~ ~ Carbonyldiimidazole o II 110 CH--C,-N_N .. --C CH. I \ \1 N'\/\/vv 'vvvvC-NH-CH __ C/ II o Fig. 2. Cyclic imide formation been distilled from P20S to remove traces of water and dimethylamine. The peptide amino groups must be protected, for example, with the tbutyloxycarbonyl group (249), which is easily removed under the conditions of the Edman degradation. Activation of the carboxyls is usually done with a several hundredfold excess carbonyldiimidazole, which should be destroyed after activation to prevent reaction and cross-linkage of resin amino groups.
0 x 10 cm Sephadex G-25 column to remove the ammonium sulfate and the 2-pyrrolidone. The optimum concentrations of enzyme and substrate will vary with the particular peptide under study, and it is useful to make a preliminary examination of the reaction progress on a small amount of the peptide before attempting to prepare a large amount for subsequent degradation or other follow-up characterization. To this end, about 50 nmol of peptide are dissolved in 100 ~l of the enzyme solution and incubated at 30°.
Advanced Methods in Protein Sequence Determination by Professor Saul B. Needleman (auth.)