Synergies between micropreparative high-performance liquid chromatography and an instrumental optical biosensor.
The recent development of an automated surface plasmon resonance technology for the measurement of biomolecular interactions (Pharmacia BIAcore) has provided new opportunities for the detection and analysis of protein-protein interactions. In the BIAcore, detection is based on changes in surface plasmon resonance which are monitored optically. Changes in surface plasmon resonance correspond to changes in surface concentration of macromolecules and can be monitored in real time. We have found that the detection sensitivity obtainable with this technology (ng/ml concentrations of specific ligands are readily detectable for many applications) is complementary "in a bidirectional manner" to micropreparative HPLC. Thus micropreparative HPLC may be used to purify and characterise reagents for the biosensor, whilst the biosensor may be used to define chromatographic parameters such as elution conditions for affinity chromatography or serve as an affinity detector for fractions obtained during chromatographic purification. Examples of such applications, including the potential of the biosensor to search for and monitor the purification of unknown ligands for which the target molecule has been identified, are shown. In particular, the use of the biosensor to monitor the purification of soluble epidermal growth factor receptor from A431 cell conditioned media is demonstrated.
Nice
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E
Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch, Royal Melbourne Hospital, Victoria, Australia.
Lackmann
M
M
Smyth
F
F
Fabri
L
L
Burgess
A W
AW
D015374:Biosensing Techniques; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D066246:ErbB Receptors / Q000302:isolation & purification; D007202:Indicators and Reagents; D055095:Optics and Photonics / Q000295:instrumentation
D007202:Indicators and Reagents; D066246:ErbB Receptors