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.*** Add Eylea and BSA ***  \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. *** Add Eylea and BSA ***