Peptide manual

We have found that although the use of an automatic synthesizer can save time and can be relatively unproblematic for simple sequences, the synthesis of peptides manually allows for more flexibility and control over the assembly process. This is particularly important for the synthesis of difficult sequences as it permits quick and easy intervention at any point. 

The initial synthesis of the EGF-like peptide carried out on a synthesizer using an Fmoc approach failed. As identified by both HPLC and MS, the resulting peptide with 48 residues was found to be a mixture containing no detectable amount of the desired peptide. Manual synthesis was then attempted. Since the... 

Kay, B.K, Winter, J., McCafferty, J. eds. Phage Display of Peptides and Proteins a Laboratory Manual. San Diego Academic Press, 1996. 

The following protocol for EPL, including purification using a CBD fusion tag followed by native chemical ligation, is based on the methods of Muir et al. (1998), Chong et al. (1997, 1998), Evans et al. (1998), Severinov and Muir (1998), and the NEB instruction manual for the IMPACT-TWIN system. The recombinant protein is recovered from the affinity column as the thioester derivative ready for reaction with a N-terminal Cys peptide or another tag containing a Cys residue. 

The synthesis can be carried out manually or automated using a Symphony/Multiplex multiple peptide synthesizer or an Argonaut Nautilus. 

Two-dimensional electrophoresis [86] is a well established technique for the separation of intact proteins. In the first dimension the proteins are separated based on their isolectric point while the second dimension separates them based on their size. The presence on the gel of the proteins is revealed by Coomassie blue or silver staining. Under favorable conditions several thousand spots can be differentiated. The gel is digitized and computer-assisted analysis of the protein spot is performed. The spots of interest are excised either manually or automatically and then digested with trypsin. Trypsin cleaves proteins at the C-terminal side of lysine and arginine. In general one spot represents one protein and the peptides are analyzed by MALDI-TOF to obtain a peptide mass fingerprint. A peptide mass fingerprint involves the determination of the masses of all pep-... 

Commonly, in vitro determination of HDAC activity is a manual assay utilizing a coupled two-step process, including enzymatic deacetylation of a substrate followed by reaction termination and readout [10]. Assays utilize nuclear extracts and substrates containing labeled (radioactive or fluorescent) acetylated histones. For the isotope-based assays, the enzymes are incubated with acetate-radiolabled histones prepared from chicken reticulocytes or chemically [ Hjacetylated peptide substrates, and the enzymatic activity is determined by liquid scintillation counting [11]. Alternatively, histones may be obtained from cells following treatment with [ H]acetyl-CoA [12]. The caveats of these approaches include the variability of prelabeled acetylated core histones within preparations, potential high costs, their labor-intensive nature and the presence of radioactive waste. 

Within the last two years since the Sequencer of Beckman Instruments became generally available, the automated method has been successfully applied predominantly to comparisons of homologous proteins such as the immunoglobulins. Elucidation of the sequence of 20 to 30 N-terminal residues in peptides as long as the L-chains, comprising 214 amino acids or the H-chains with more than 400 residues would have been impossible to achieve by the manual procedure without prior cleavage of the molecules and isolation of the N-terminal regions. 

See Section 3 in the Instruction Manual of the Beckman Protein Peptide Sequencer. Palo Alto, Calif. Spinco Division of Beckman Instruments Inc. 1970. 

The sequence of solid-phase assembly and cyclization reactions used to prepare the bis(Ampa)-bridged peptide 31 is summarized in Scheme 19. All solid-phase procedures were performed in a standard, custom-built, 200-mL capacity, manual peptide synthesis reaction vessel. 165 ... 

A wide choice of peptide synthesizers is currently available, ranging from manual to fully automated. They are all based on solid-phase peptide synthesis methodologies in which either f-butoxy carbonyl (t-boc) (11), or 9-fluor-enylmethoxycarbonyl (Fmoc) (12) is the major protecting group during synthesis. A detailed description of peptide synthesis is clearly beyond the scope of this chapter, and further information on practical and theoretical approaches to this chemistry may be found elsewhere (13-15). However, a brief outline of solid-phase synthesis may prove useful. 

Solid-phase synthesis of the peptide was carried out manually on a Rink amide resin (435 mg, substitution level 0.46 mmol -g 1) applying the Fmoc strategy. After removal of the Fmoc group attached to the resin with 20% piperidine in DMF (20 min), Fmoc-Tyr(tBu)-OH (230 mg, 0.5 mmol) was introduced with DIC (78 pL, 0.5 mmol) in the presence of HOBt (68 mg, 0.5 mmol) (reaction time 2h). After... 

Manual Stepwise Solid-Phase Peptide Synthesis General Procedure 177 791... 

---