Protocol

Protein Separation on Ceramic Hydroxyapatite Using High-Performance Liquid Chromatography

This protocol was adapted from "Protein Chromatography on Hydroxyapatite," contributed by Richard J. Simpson, Chapter 13, 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

Ceramic hydroxyapatite is a spherical, macroporous form of hydroxyapatite that can be used reproducibly at high flow rates and high pressures in large columns. This protocol, adapted with permission from the Bio-Rad application notes for CHT ceramic hydroxyapatite, describes the separation of proteins using ceramic hydroxyapatite and HPLC.

MATERIALS

IMPORTANT:

Use carbonate-free H₂O to prepare all solutions. Carbonate reacts with calcium ions to form a crust of calcium carbonate at the top of the column.

Reagents

Hydroxyapatite buffer A

Hydroxyapatite buffer B

Hydroxyapatite column storage buffer

Ethanol, methanol, isopropanol, or acetonitrile

KOH (or NaOH) (1-2 M)

Potassium phosphate (or sodium phosphate) . . . [Full Text of this Article]

Equipment

METHOD

TROUBLESHOOTING