Progress Toward Polybrene Purification and Utility in Micro-Peptide/Protein Sequencing

Biomedical Chromatography

Fabri

Hammacher

et al. 1993

Data on the characterization of anion and cation micropreparative (50 x 1.6 mm i.d.) HPLC columns is presented. It is shown how subnanomole quantities of protein can be efficiently recovered from such columns, rendering them compatible for use in multidimensional chromatographic strategies for the purification of trace biological samples. By selection of appropriate solvent systems (e.g., buffer-free sodium chloride solutions), the small eluant peak volumes can be loaded directly onto the gas phase sequencer, and N-terminal sequence data obtained. The potential of the technique is illustrated for the purification of a GTPase activating protein (GAP-3).

Section: N-terminal Sequence Analysis From Ion Exchange Columnsmentioning

confidence: 98%

Section: N-terminal Sequence Analysis From Ion Exchange Columnsmentioning

confidence: 99%

Micropreparative ion exchange high performance liquid chromatography: Applications to microsequence analysis

Biomedical Chromatography

Fabri

Hammacher

et al. 1993

Micropreparative high performance liquid chromatography of proteins and peptides: Principles and applications

1996

Biopolymers

The role of micropreparative high performance liquid chromatography columns for the efficient purification of microgram to nanogram quantities of proteins and peptides in small eluent volumes at high concentration is described. The historical development of the technique with respect to column dimensions and instrumentation is outlined. The potential of this technique to prepare samples for use with highly specific and sensitive analytical techniques (e.g., N‐terminal sequence analysis, mass spectrometry, biosensor analysis, biological assay) or for the recovery of low levels of material following chemical modification (e.g., radio iodination, enzymatic semisynthesis, chemical conjugation) is illustrated for a number of proteins including growth factors and their receptors. © 1996 John Wiley & Sons, Inc.

High‐Performance Liquid Chromatographic Separations and Equipment in Peptide and Protein Analysis, Miniaturization of

Encyclopedia of Analytical Chemistry

Catimel

2000

The use of short narrow‐bore (2.1‐mm i.d.), microbore (1‐mm i.d.) or capillary (<1‐mm i.d.) columns allows the recovery of purified proteins and peptides in reduced volume at concomitantly increased concentration compared with larger bore columns. Additionally, sample detection is correspondingly improved for concentration‐dependent detectors. Sample volumes are not limiting: large sample volumes can be loaded on to interactive supports (e.g. reversed‐phase (RP), ion‐exchange (IEX), hydrophobic interaction (HI), affinity) under appropriate mobile phase conditions (trace enrichment). Suitable instrumentation requires careful consideration with regard to minimized pre‐ and postcolumn dead volumes, accurate gradient formation at low flow rates, detector flow cell geometry and sample recovery. These techniques are ideally suited for the purification of trace protein or peptide components in bulk biological samples prior to application to highly specific and sensitive downstream analytical techniques (e.g. microsequence analysis, mass spectrometry (MS), biosensor analysis, biological assay). They also facilitate the recovery of low levels of material following chemical or enzymatic modification (e.g. radioiodination, enzymatic semi‐synthesis, chemical derivatization). The techniques can also be applied to the development of large‐scale processes, whereby multidimensional purification protocols can be rapidly developed using minimum amounts of sample, solvent and column packing materials and then scaled up appropriately.