N-Terminal Protein Sequencer

PVDF membranes bind proteins primarily through hydrophobic interactions and are commonly used for their chemical resistance as well as physical stability. High-affinity PVDF membranes such as Trans-Blot (Bio-Rad), ProBlott (Perkin-Elmer), and Immobilon-PSQ (Mil-lipore) are preferred for blots intended for use in N-terminal protein sequencing, whereas low-retention membranes such as Immobilon-P (Millipore) may produce lower background in both immunoblotting and common staining procedures. In addition, low-retention membranes are preferred when proteins will be extracted from the membrane. 

The sample application is compatible with diverse samples recovered in various buffers (phosphate, inorganic salts, HEPES) and solvents (HPLC fractions). Samples that have been subjected to amino-terminal sequence analysis using the HP G1005A N-terminal protein sequencing system and Zitex as a reaction support may be transferred to C-terminal sequencer columns and subjected to C-terminal sequence analysis with the HP G1009A C-terminal sequencing system. 

Comparison of the High Sensitivity and Standard Versions of Applied Biosystems Procise 494 N-Terminal Protein Sequencers using Various Sequencing Supports... 

Positive Identification of Glycosylation Sites In Proteins And Peptides Using A Modified Beckman LF 3600 N-Terminal Protein Sequencer... 

The techniques developed for the LF 3600 N-terminal protein sequencer (Beckman Instr.) provide a fast and efficient way to positively identify Ser (0-linked Sac), -Thr (0-linked Sac) and Asn (N-linked Sac) carbohydrate structures during N-terminal sequence analysis (Gooley et al., 1995). Liquid phase anhydrous trifluoroacetic acid (TEA) is used to extract glycosylated, polar amino acid derivatives from the reaction cartridge. These amino acids are then converted into PTH derivatives which can be... 

Smith, B. J., SDS-polyacrylamide gel electrophoresis for N-terminal protein sequencing, in Protein Sequencing Protocols, Smith, B. J., Ed., Humana Press, Totowa, NJ, chap. 2, 1997, 17-24. 

The derived amino acid sequences (shown as cDNA) from the cDNAs are given together with the N-terminal protein sequence of the pea protein (shown as Prot). The proposed Fe-S binding domains are shown boxed and an N-terminal hydrophobic domain is underlined. The arrow indicates the processing site between the mature protein and the presequence. 

For routine determination of unknown N-terminal protein sequences, at least 50-100 pmole of the purified protein should be available. The only method for the accurate quantitation of low picomole amounts of protein is quantitative amino acid composition analysis. Twenty to fifty picomoles of protein should be set aside... 

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