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 whilst the manuscript for the 1st a part of this e-book used to be proposed, it was once expected that the dialogue of the whole box of protein se quencing should be lined in one quantity - from purification and characterization of the protein via fragmentation by means of chemical or enzymic skill and, ultimately, to reassembly of the pointed out person peptides into the reconstructed overall series. It quickly turned obvious that this might now not be attainable. whereas the rationale was once to limit the disclose of strategies basically to that info fascinated by "hands on" rainy chemistry, it grew to become obvious thorough presentation will require, additionally, a dialogue of yes instrumental and extra theoretical techniques no longer incorporated within the first quantity. additionally, the full knowing of the sector of protein sequencing has complex noticeably because the inception of this publication. the aim of the 1st quantity was once to supply useful details in adequate element to allow the researcher to adopt the particular sequencing tactics in his personal laboratory.
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Extra resources for Advanced Methods in Protein Sequence Determination
However, when a peptide is activated with p-phenylene diisothiocyanate, the peptide amino groups acquire reactive hydrophobic "heads" which can readily penetrate and react with the hydrophobic amino resin. Carboxyl-activated peptides, however, are generally polar and cannot penetrate as easily. Also arylamines are not particularly good nucleophiles in carbonyl reactions, and react rather slowly. On the other hand, triethylenetetramine (TETA) resin, with its long polyamine side chains, is hydrophilic, as seen by its swelling properties in polar solvents such as methanol.
1 M 2-pyrrolidone as an enzyme stabilizer (21). Care must be taken to maintain the temperature of the suspension during sonication in the range 0-10 0 . After sonication the material is centrifuged at 39,000 g at 00 for 3060 min. The supernatant liquid containing the enzyme activity is carefully decanted, and if necessary, it should be centrifuged a second time. The A280 of the supernatant should be at least 50; if it is higher, it can be diluted with the phosphate buffer described above. Nucleic acids may be precipitated by adding one volume of 1% protamine sulfate to six volumes of cold supernatant containing the enzyme.
The material is then dialyzed against the same buffer to remove the residual ammonium sulfate and 2-pyrrolidone (the stabilizer is also an inhibitor). Usually three successive 1 h dialyses against 250 ml buffer each are sufficient. 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.
Advanced Methods in Protein Sequence Determination by Professor Saul B. Needleman (auth.)