Applied Biosystems Model

Protein Sequencing. The xylanase protein sequence was obtained by stepwise automated E an degradation ter an initial double-coupling protocol, using an Applied Biosystems model 470A gas-phase sequenator equipped with an on-line Applied Biosystems model 120A PTH-amino acid analyzer 13). 

Transport peptides can be synthesized using either t-Boc or Fmoc solid phase peptide synthesis strategies with a synthesizer or manually. We routinely synthesize CPPs in a stepwise manner on solid support using an Applied Biosystems Model 431A peptide synthesizer. tert-Butyloxycarbonyl amino acids are coupled as 1-hydroxybenzotriazole (HOBt) esters to a p-methylbenzylhydryl-amine (MBHA) resin (65). C-terminally amidated CPPs are less prone to degradation and show higher internalization efficiency than carboxylic acid derivatives. 

The protein sequencer used for this study was an Applied Biosystems Model 477A, used without the Model 120A PTH analyzer, liie sequencer was fitted with an Applied Biosystems Micro cartridge, but all other hardware was as supplied by the maniifacturer. Reaction and conversion cycles were modified to accommodate this configuration. Some modified solvents and reagents were used and will be discussed below. 

Applied Biosystems, Inc. (1989) Applied Biosystems Model 477A Protein-Peptide Sequencing System Users Manual, Foster City, CA. 

The quantitative ninhydrin test has not been automated. The Applied Biosystems Models 430, 431, and 433 Peptide Synthesizers, however, have the capability to automatically remove 3-10 mg resin samples after each coupling for a manual ninhydrin test. 

In the case of those peptides selected (on the basis of the combined criteria of absorbance peak shape and MALDI-MS spectrum) for sequencing, the appropriate fraction was loaded directly onto an Applied Biosystems Model 470, 477 or HS-Procise instrument operated according to the manufacturer s recommended protocol and as described previously in more detail (5). Immediately following sequencing, all peptide sequences were searched via the "Blast" email server operated by the National Center for Biotechnology Information (9),... 

Peptides were sequenced using either an Applied Biosystems Model 470 or 477 or a Hewlett Packard GIOOOA protein sequencer, each equipped with narrow bore RP-HPLC for on-line analysis of the PTH-amino acids. Mass analysis of peptides was performed using matrix-assisted laser desorption/ionization mass spectrometry on a KRATOS MALDl 111 with a-cyano-4 hydroxy-cinnamic acid as the matrix. 

All separations were performed on an Applied Biosystems Model 270A-HT automated capillary electrophoresis system. All separation media were prepared using Milli-Q water and filtered through 0.22- m filters obtained from Gelman Sciences... 

Liquid chromatography mass spectrometry (LCMS) may be performed with a PE Sciex API 300 electrospray triple-quadrupole mass spectrometer, and an Applied Biosystems model 130 HPLC system with a 5 x Vydac C18 column (1 x 250 mm, using aqueous tnfluoroacetic acid/acetonitrile solvents), or equivalent... 

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