Biotinylation a fast and reproducible method for labelling Trypanosoma cruzi cell surface proteins.
In this study we describe a simple and rapid method that uses sulfo-N-hydroxy-succinimidobiotin (sulfo-NHS-biotin) to label Trypanosoma cruzi surface proteins stably without significant loss of biological function. Efficient labelling can be obtained with as little as a 5 minute incubation of parasites in an appropriate concentration of sulfo-NHS-biotin at 4 degrees C. After labelling under these conditions, biotinylated parasites exhibited levels of motility, viability, and in vivo infectivity comparable to those seen with unlabelled control parasites. Moreover, the biological activity of T-DAF, a complement regulatory protein found on the parasite surface, was unaffected when biotinylated under these conditions. Biotinylated surface proteins can be easily detected in a variety of non-radioactive assays employing conjugated streptavidin as a developer. Compared to alternative techniques of surface labelling described in the literature, this method offers better preservation of biological function as well as greater ease of use and safety.
Tambourgi
D V
DV
Instituto de Ciências Biomédicas da Universidade de São Paulo, Brazil.
Ogden
G B
GB
Hall
B F
BF
da Silva
W D
WD
Kipnis
T L
TL
D000345:Affinity Labels; D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis / Q000302:isolation & purification; D001710:Biotin / Q000031:analogs & derivatives* / Q000494:pharmacology; D015153:Blotting, Western; D018958:CD55 Antigens; D003169:Complement Inactivator Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000302:isolation & purification; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry*; D051379:Mice; D015800:Protozoan Proteins / Q000096:biosynthesis / Q000737:chemistry*; D013388:Succinimides / Q000494:pharmacology; D013997:Time Factors; D014349:Trypanosoma cruzi / Q000737:chemistry / Q000187:drug effects* / Q000502:physiology
D000345:Affinity Labels; D015703:Antigens, CD; D018958:CD55 Antigens; D003169:Complement Inactivator Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D015800:Protozoan Proteins; D013388:Succinimides; C065130:sulfo-N-hydroxysuccinimide-biotin; D001710:Biotin