American Society of Hematology

Fig. 5.

$Fig. 5. Binding of biotinylated platelets to alboaggregin A. / Biotinylated platelet proteins eluted from an alboaggregin A affinity column with increasing amounts of SDS were separated by SDS-PAGE (7%-17% acrylamide gradient) and transferred to PVDF membranes. (A) Membranes were incubated with avidin-coupled alkaline phosphatase and detected with BCIP and NBT. (B) Membranes were incubated with anti-GPVI Ab, anti-GPIbβ (framed bands), and a peroxidase-conjugated second Ab and detected by using chemiluminescence. (C) Membranes were incubated with avidin-coupled alkaline phosphatase and stained with BCIP and NBT. (D) Membranes were incubated with anti-GPIb Ab, anti-GPIX (framed bands), and a peroxidase-conjugated second Ab and stained by using chemiluminescence. In each, lane 1 is the WGA-bound fraction of Triton X-100 phase of biotinylated platelet glycoproteins (starting material); lane 2, the flow-through fraction; lane 3, the first fraction eluted with 0.08% SDS; lane 4, the second fraction eluted with 0.08% SDS; lane 5, the fraction eluted with 0.2% SDS; and lane 6, the immunoprecipitation of starting material with (A) anti-GPVI Abs and (C) anti-GPIb Abs.$

Binding of biotinylated platelets to alboaggregin A.

Biotinylated platelet proteins eluted from an alboaggregin A affinity column with increasing amounts of SDS were separated by SDS-PAGE (7%-17% acrylamide gradient) and transferred to PVDF membranes. (A) Membranes were incubated with avidin-coupled alkaline phosphatase and detected with BCIP and NBT. (B) Membranes were incubated with anti-GPVI Ab, anti-GPIbβ (framed bands), and a peroxidase-conjugated second Ab and detected by using chemiluminescence. (C) Membranes were incubated with avidin-coupled alkaline phosphatase and stained with BCIP and NBT. (D) Membranes were incubated with anti-GPIb Ab, anti-GPIX (framed bands), and a peroxidase-conjugated second Ab and stained by using chemiluminescence. In each, lane 1 is the WGA-bound fraction of Triton X-100 phase of biotinylated platelet glycoproteins (starting material); lane 2, the flow-through fraction; lane 3, the first fraction eluted with 0.08% SDS; lane 4, the second fraction eluted with 0.08% SDS; lane 5, the fraction eluted with 0.2% SDS; and lane 6, the immunoprecipitation of starting material with (A) anti-GPVI Abs and (C) anti-GPIb Abs.

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