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Introduction to Modern Liquid Chromatography, Third Edition part 66. High-performance liquid chromatography (HPLC) is today the leading technique for chemical analysis and related applications, with an ability to separate, analyze, and/or purify virtually any sample. Snyder and Kirkland's Introduction to Modern Liquid Chromatography has long represented the premier reference to HPLC. This Third Edition, with John Dolan as added coauthor, addresses important improvements in columns and equipment, as well as major advances in our understanding of HPLC separation, our ability to solve problems that were troublesome in the past, and the application of HPLC for new kinds of samples. . | 606 BIOCHEMICAL AND SYNTHETIC POLYMER SEPARATIONS matrix Initial column chelator Step 1. load metal Step 3 Desorb protein Step 2. Bind protein P EDTA Step 4 Regenerate column Cu EDTA Figure I3.I9 Steps in the use of IMAC. Adapted from 56 . amino-acid side chains in the protein can form coordination complexes with metals so IMAC is a general method for protein separation. The primary interaction in IMAC is with the imidazole group of histidine in its unprotonated form 58 the strength ofmetalbin dinghy different amino-acid groom in the protein molecule decreases in the following order his yrp tyr dPhe agg metgigly Cystci ncrcsidi.ies caobindmctals buttheymay notbeavailable on the protein surface in theeeduced shete sincethoyreadily oxidize in the presence of metal ions 59 . Cysteine-containing proteins may therefore require a reducing environment addition of 2-mercaptoethanol sir ditlaiothrcitol m orelcr to seaintrm the cysteine residues in their active -SH form. Aromatic residues can contribute indirectly to retention by enhcncmg het bindieg of nciyhbesmghitsidinse 59li The ahmeieanceoribaekbne tending th CMAC columns hns bepn tepleiocd by genetically ehctnedrmgpein llsriPhl eendesces mlotargclpaolc es foaenre nf purificatioe.Atter petfetymreebioding dlution aodrecovery ofthr twy e naetnin the polyhistidine sequsnce eanbe cleavtdSy meme of edrCnxypentiOnsr A 59 . Phosphoprotemsdngphosenontptihef dinnsetedtivelgeodMAC colernaichelated with Fe 3 and Ge tiyndnMAC has bneome akeo twlm charynterieing Ae pliospliopgyctHnc S . 13.4 SEPARATION OF PEPTIDES AND PROTEINS 607 The high capacity of IMAC columns and the high recovery of protein mass and activity make this technique useful for preparative and process-scale chromatography. For protein purification IMAC compares favorably with affinity chromatography in terms of binding strength and capacity and has the advantages of stability over a wide range of conditions and use-cycles relatively mild elution conditions .
