Cite as: Cold Spring Harb. Protoc.; 2006; doi:10.1101/pdb.prot4200

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Standard Chromatographic Conditions for RP-HPLC of Proteins

C. David Carr and Robert L. Moritz

This protocol was adapted from "Role of Reversed-Phase High-Performance Liquid Chromatography in Protein Isolation and Purification," contributed by C. David Carr and Robert L. Moritz, Chapter 8, in Purifying Proteins for Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2004.

The first 100 words of the full text of this article appear below.


INTRODUCTION

Because of its very high resolving power, reversed-phase high-performance liquid chromatography (RP-HPLC) plays a central role in fractionation of complex polypeptide samples during protein (or peptide) purification and identification, especially prior to mass spectrometry. Before analyzing a mixture of proteins or peptides by RP-HPLC, it is essential to evaluate the HPLC system. When a new column arrives in the laboratory, read the manufacturer’s instructions for proper conditioning, use, and storage. Once these are understood, carry out this protocol to perform a test separation using a set of protein standards that have been designed to evaluate column performance.


MATERIALS

Reagents

Peptide mixture generated . . . [Full Text of this Article]

Equipment


METHOD


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