Isolation and purification of chemokines from natural sources.
Chemokines (e.g., IL-8) were originally identified as chemotactic proteins obtained from various different natural sources. Today, using the genome walking strategy an ever-increasing number of novel genes encoding chemokines have been discovered that were expressed in bacteria or eukaryotic cells and subsequently tested for biological activity. Usually biological significance of the considered chemokine is extrapolated from these data. The increasing evidence, however, that post-translational modification of chemokines can dramatically affect its biological activity makes it necessary to identify the naturally occurring chemokines in order to identify its biological function. Furthermore, with the isolation of natural chemokines, evidence is provided that transcription of chemokine genes is really followed by translation into a bioactive molecule. Purification of chemokines from natural sources requires special strategies: The bioassay or immunoassay should allow screening of high-performance liquid chromatography fractions and detection of the required chemokine at low concentration. Parameters that affect the detection of bioactivity and immunoreactivity (giving either false positive or false negative results) should be carefully considered. In this article methods for molecular characterization of chemokines from both cell culture supernatants and human tissue (lesional inflammatory skin scales) will be described.
Schröder
J M
JM
Department of Dermatology, University of Kiel, D-24105 Kiel, Germany. jschroeder@dermatology.uni-kiel.de
D001671:Biochemistry / Q000379:methods*; D002478:Cells, Cultured; D018925:Chemokines / Q000302:isolation & purification*; D002633:Chemotaxis; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004804:Eosinophils / Q000378:metabolism; D006801:Humans; D009504:Neutrophils / Q000378:metabolism; D012867:Skin / Q000378:metabolism
D018925:Chemokines