Purification peptides

E. Rothsteia, ia R. Harrison, ed.. Protein and Peptide Purification Process Eepelopment and Scale-Ep, Marcel Dekker, Inc., New York, ia press. 

Purify the tryptic peptides by chromatography on a C18 column to remove salts (follow the manufacturer s directions for peptide purification). Dry the eluent and redissolve the peptides in 20 pi 0.1 percent formic acid. 

Following cleavage with hydrogen fluoride, the various classes of peptides were separated in a one-step purification procedure on a tertiary or quaternary amine column. After removal of the Sulfmoc group with 5% TEA, homogeneous Leu-Ala-Gly-Val, for example, was obtained. The Sulfmoc procedure was also very effective for purification of synthetic thymosin oq (28 residues). 87 This was the first use of an Fmoc derivative for selective and reversible orthogonal peptide purification. 

Matsuidaira, P. T., ed. (1995) A Practical Guide to Protein and Peptide Purification for Microsequencing, Academic Press, San Diego, CA... 

Key Words Invertebrate immunity antimicrobial peptides immune effectors mass spectrometry drug discovery Drosophila arthropods peptide purification molecular mass fingerprints bioactive peptides. 

The main goal of this chapter is to describe the synthesis details of complex, orthogonally protected peptide constructs. Thus, major emphasis is placed on the peptide chain assembly design and practice and the alterations from the solid-phase synthesis of simple, nonmodified peptides. The technology for peptide purification and quality control is not significantly different from that of other peptides, and these methods will be just briefly described. Many chapters of this book focus on the optimization of HPLC and MALDI-MS procedures for peptide separation and analysis and illustrate the expected and/or acceptable quality control parameters. 

A. Fusion partners for recombinant protein/peptide purification ... 

The simplest and often the most cost effective way to combat friction is to reduce flow rate to a minimum. By no coincidence, this often leads to an increase in the efficiency of a separation since in many circumstances for preparative purifications, the less experienced have followed a linear scale-up from analytical column flow rates. In an ideal world each separation should, at some stage, involve a flow rate optimization. The fundamental principles behind this are discussed by JJ van Deemter[52 in what is probably the most cited paper in the history of chromatography. In summary, this suggests doing a graphical plot of separation efficiency versus flow rate and is particularly important for peptide purification where mass transport is comparatively slow. The van Deemter equation in simplified form can be represented as ... 

The eluents most commonly used in peptide purification by reversed phase HPLC are water and acetonitrile. These are often buffered with trifluoroacetic acid (0.1% v/v, TFA), ammonium acetate (0.05-0.1 mol/dm3 at pH 4-8) or phosphate (0.05-0.1 mol/dm3 sodium or potassium salt at pH 2-8). In addition, polymeric reversed phase media also performs well at high pH and is often buffered with ammonium hydroxide or ammonium bicarbonate (0.05-0.1 mol/dm3 at pH 8-9). 

Peptide Purification At pH 7.0, in what order would the following three peptides be eluted from a column filled with a cation-exchange polymer Their amino acid compositions are ... 

Electrophoretic techniques, such as two-dimensional electrophoresis or isoelectric focusing, lend themselves under appropriate conditions to the separation with excellent resolution of small amounts of samples. They have mainly found use as powerful methods of analysis since their general applicability in the areas of peptide purification and isolation has, until recently, been severely restricted by limitations in sample capacity and instrumental design. 

Stainless steel columns packed with a suitable stationary phase are used for peptide purification by HPLC. Reverse phase (RP) chromatography utilizes a stationary phase that is a nonpolar compound such as Cig hydrocarbon covalently bound to porous silica. For the purification of more hydrophobic peptides, C4- or Cg-RP columns have also been used. 

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