Receptor-ligand interactions measured by an improved spun column chromatography technique. A high efficiency and high throughput size separation method.
Size exclusion chromatography may under the right circumstances be an easy and powerful way to measure in solution the interaction between a receptor an dits ligand. Spun column chromatography is a fast size exclusion technique of increasing popularity, however, little information exists on the method development essential to obtain efficient separation in particular when used for analytical purposes. In this paper we describe a systematic approach to select the optimal parameters for spun column separation including a simple modification of the technique whereby the spun columns are eluted by high-speed gradient centrifugation. This modification is easy to implement and it considerably improves spun column performance. We hypothesize that the high-speed centrifugation step leads to the release of additional buffer which assists in the complete elution of excluded molecules while the gradient centrifugation helps to achieve equilibrium across the gel matrix during the elution. The new method has been used successfully for several different receptor-ligand interactions, and this paper describes a general approach on how to develop new applications of the technique.
Buus
S
S
Institute of Medical Microbiology and Immunology, Medical Faculty, University of Copenhagen, Panum Institute, Denmark.
Stryhn
A
A
Winther
K
K
Kirkby
N
N
Pedersen
L O
LO
D000595:Amino Acid Sequence; D000818:Animals; D002845:Chromatography / Q000379:methods*; D002850:Chromatography, Gel / Q000379:methods*; D003911:Dextrans; D005782:Gels; D006389:Hemagglutinins, Viral / Q000737:chemistry / Q000378:metabolism; D015395:Histocompatibility Antigens Class I / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009698:Nucleoproteins / Q000737:chemistry / Q000378:metabolism; D009975:Orthomyxoviridae / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism
D003911:Dextrans; D005782:Gels; D006389:Hemagglutinins, Viral; D015395:Histocompatibility Antigens Class I; D009698:Nucleoproteins; D010446:Peptide Fragments; C025614:sephadex