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Liisa Hirvonen edited Method.tex
over 8 years ago
Commit id: aea0fc8a8c34b0ee1e504162ce3cef15478f900c
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\subsection{Reagents}
Ranibizumab was purchased from Novartis (Frimley, UK)
(Lucentis (Lucentis\textregistered 10~mg/ml) and bevacizumab from Roche (Welwyn Garden City, UK)
(Avastin (Avastin\textregistered 25~mg/ml).
BSA was purchased from Sigma (Poole, UK) at the highest available purity. Aflibercept was purchased from Bayer plc (Newbury, UK) as a 40~mg/ml solution (Eylea\textregistered). All other reagents were of the highest available purity and were either from Sigma (Poole, UK), VWR (Lutterworth, UK), or Merck (Hoddesdon, UK) unless otherwise specified.
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\subsection{Sample preparation}
Ranibizumab and bevacizumab All protein solutions were
dialysed diluted with sterile phosphate buffered saline (PBS; Sigma; pH~7.4) to 5mg/mL and then dialyzed twice against phosphate buffered saline (PBS) pH 7.4 using D-Tube Midi Dialyzer units from Novagen (6-8~kD cut-off). Subsequently, the purity of the dialysed proteins was
checked confirmed by standard SDS-PAGE. The proteins were then conjugated to the
dye ruthenium dye. This was achieved by reacting the succinimidyl ester-modified dye, Bis(2,2′-bipyridine)-4′-methyl-4-carboxybipyridine-ruthenium N-succinimidyl ester-bis(hexafluorophosphate) (synonym, Ru(bpy)$_2$(mcbpy-O-Su-ester)(PF$_6$)$_2$, Sigma-Aldrich) by using a succinimidyl ester-modified fluorophore with a short linker (Invitrogen, F6130). Conjugation reactions were performed in PBS adjusted with bicine buffer to pH 8.6 at 2~mg/ml protein concentration with the activated fluorophore ester being used in excess. The reaction was stopped after two hours and conjugated proteins were separated from remaining free dye by size exclusion chromatography
(7~kD cut-off; Thermo Fisher). (two times via 7~kD cut-off Zeba\textregistered spin columns (Thermo Fisher, UK)). Using this method we generated ranibizumab conjugated to dye with a dye:protein ratio of 1.1:1 and bevacizumab with a dye:protein ratio of 1.3:1. Azide (2.0~mM) was added to all dye-conjugated drugs to protect them from microbial deterioration.
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