Reversed-phase High Performance Liquid Chromatography of proteins

Current Protocols in Protein Science
Djuro Josic, Spomenka Kovac

Abstract

Reversed-phase HPLC (RP-HPLC) is one of most important techniques for protein separations and the method of choice for peptide separation. RP-HPLC has been applied on the nano, micro, and analytical scale, and has also been scaled up for preparative purifications, to large industrial scale. Because of its compatibility with mass spectrometry, RP-HPLC is an indispensable tool in proteomic research. With modern instrumentation and columns, complex mixtures of peptides and proteins can be separated at attomolar levels for further analysis. In addition, preparative RP-HPLC is often used for large-scale purification of proteins. This unit provides protocols for packing and testing a column, protein separation by use of gradient or step elution, desalting of protein solutions, and separation of enzymatic digests before mass spectrometric analyses. A protocol is also provided for cleaning, regenerating, and storing reversed-phase chromatography columns.

References

Jan 1, 1984·Methods in Enzymology·M T Hearn
Jun 23, 1995·Journal of Chromatography. a·H Chen, C Horváth
Mar 15, 1997·Analytical Chemistry·J E MacNairJ W Jorgenson
Sep 19, 2002·Journal of Chromatography. a·John W Dolan
Nov 17, 2004·Journal of Chromatography. a·Yang ShiJames A Wilkins
Mar 1, 2006·Analytical Chemistry·John W Eschelbach, James W Jorgenson
Jun 3, 2006·Journal of Proteome Research·James MartosellaBarry E Boyes
Jun 24, 2006·Analytical Chemistry·Frantisek Svec, Christian G Huber
Dec 19, 2006·Journal of Chromatography. a·Djuro Josic, James G Clifton
May 23, 2008·Biotechnology and Bioengineering·Jonathan L CoffmanBrian D Kelley

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Citations

Dec 3, 2015·Current Protocols in Chemical Biology·Hosahalli P HemanthaAshraf Brik
Feb 9, 2019·BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology·Anjali Arora, Kumaravel Somasundaram
Jan 22, 2015·Current Protocols in Nucleic Acid Chemistry·Yosuke TaniguchiShigeki Sasaki
Jan 31, 2020·Expert Review of Proteomics·Esei T TeclemariamStephanie M Cologna
Feb 6, 2017·Current Protocols in Protein Science·K Ilker SenHirsh Nanda
Jul 15, 2021·International Journal of Microbiology·Ellen TwomeyMáire Begley
Aug 2, 2017·Current Protocols in Protein Science·John M AntosHidde L Ploegh

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