实验方案
操作步骤
1.运行Antibody-Buccutite MTA反应
1.1将抗体溶液直接加入到Buccutite MTA(组分B)的小瓶中,并通过反复移液几次将其充分混合或将小瓶涡旋几秒钟。
1.2将抗体-Buccutite MTA反应混合物在室温下保持30-60分钟。 注意:如果需要,抗体-Buccutite MTA反应混合物可以旋转或摇动更长时间。
2.制备抗体-APC-Cy5.5缀合物
2.1通过向Buccutite FOL活化的APC-Cy5.5(组分A)的小瓶中加入50μLddH2O制备Buccutite FOL活化的APC-Cy5.5溶液,通过反复移液几次充分混合或将小瓶涡旋几秒钟。
2.2将整瓶Buccutite FOL活化的APC-Cy5.5溶液混合到纯化的抗体-Buccutite MTA溶液(来自步骤纯化抗体-Buccutite MTA溶液)中,充分混合并在室温下旋转混合物1小时。
2.3抗体-APC-Cy5.5结合物现在可以使用了。 注意:立即使用时,抗体-APC-Cy5.5结合物需要用您选择的缓冲液稀释。 注意:对于长期储存,需要浓缩或冷冻干燥抗体-APC-Cy5.5结合物溶液。
3.抗体-APC-Cy5.5共轭物的储存
抗体缀合物应在载体抗体(例如0.1%牛血清白蛋白)存在下以> 0.5mg / mL储存。 当在2mM叠氮化钠存在下储存并避光时,抗体-APC-Cy5.5缀合物溶液可以在4℃下储存两个月而没有显着变化。 对于更长时间的储存,可以将抗体-APC-Cy5.5缀合物冻干并在≤-20℃下储存。
参考文献
Chromophore attachment to phycobiliprotein beta-subunits: phycocyanobilin:cysteine-beta84 phycobiliprotein lyase activity of CpeS-like protein from Anabaena Sp. PCC7120
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Journal: J Biol Chem (2006): 8573
Excitation energy transfer from phycobiliprotein to chlorophyll d in intact cells of Acaryochloris marina studied by time- and wavelength-resolved fluorescence spectroscopy
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Journal: Microbiology (2001): 3171
Novel activity of a phycobiliprotein lyase: both the attachment of phycocyanobilin and the isomerization to phycoviolobilin are catalyzed by the proteins PecE and PecF encoded by the phycoerythrocyanin operon
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[Phycobiliprotein and fluorescence immunological assay]
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