First synthetic probe for the detection and quantification of a protein with a potential alpha,(2-->8)sialyltransferase activity.
There is considerable interest in monitoring alpha,(2-->8)sialyltransferase (ST8) levels; however, there are few specific and sensitive methods to directly detect and quantitate the protein. This paper reports the development of a synthetic probe composed of oxidized colominic acid coupled to biotinyl-L-lysine hydrazide to detect and quantify ST8 with putative "initiase" activity and its use in three solid-phase applications. The detection limit observed for ST8 purified from K562 cells was approximately 2 pg by dot-blot analysis. In Western blots the probe bound and specifically recognized a protein band corresponding to ST8. In ELISA a linear dose response was obtained for pure protein in the range of 50-200 pg. Analysis of 3'-azido-3'deoxythymidine-treated cells by all three methods showed a reduction in ST8 compared to control cells; treated cells had 73% of control levels by ELISA. This probe will be useful for studies on the expression ST8 and its role in glycoconjugate biosynthesis.
D'Andrea
Gabriele
G
Department of Biomedical Sciences and Technologies, University of L'Aquila, Coppito 2, Via Vetoio, I-67100 L'Aquila, Italy. gadan@cc.univaq.it
Lizzi
Anna Rita
AR
Oratore
Arduino
A
D045744:Cell Line, Tumor; D006801:Humans; D015335:Molecular Probes / Q000032:analysis* / Q000138:chemical synthesis; D011134:Polysaccharides / Q000032:analysis / Q000138:chemical synthesis; D011506:Proteins / Q000032:analysis*; D012799:Sialyltransferases / Q000032:analysis*
D015335:Molecular Probes; D011134:Polysaccharides; D011506:Proteins; C014420:colominic acid; D012799:Sialyltransferases