Use of fluorescamine as an effective blocking reagent to reduce the background in protein sequence analyses by the beckman automated sequencer
✍ Scribed by Ajit S. Bhown; J.Claude Bennett; Paul H. Morgan; John E. Mole
- Publisher
- Elsevier Science
- Year
- 1981
- Tongue
- English
- Weight
- 404 KB
- Volume
- 112
- Category
- Article
- ISSN
- 0003-2697
No coin nor oath required. For personal study only.
✦ Synopsis
As an effective aid to further develop the strategies for extended amino acid sequence determinations, the use of fluorescamine (fluram) as a novel blocking reagent to chemically reduce the newly generated amino termini responsible for the progressively increasing background is described. The method involves interruption of the run when proline has been determined to be the amino terminus, deletion of PITC delivery, in situ treatment of the sample with fluram within the spinning cup extraction of the reaction products using an ethyl acetate:benzene mixture, HFBA delivery, and second extraction with 1-chlorobutane. The normal program is then allowed to continue until the next proline is reached and the entire blocking procedure is repeated. Use of this multiple-sequencer-run approach can provide extended sequence information on nanomole amounts of proteins. The efficiency of the method has been demonstrated by direct comparison of the results of the sequence analysis of amyloid P-component with (63 residues) and without (46 residues) fluram treatment.