For imaging and biodistribution and cell binding studies, the diabody was labelled with 99mTc-tricarbonyl ([99mTc(CO)3]+) via the C-terminal (His)6-tag. The IsoLink kit (Covidien, Petten, The Netherlands) was used to convert 2,200 to 2,500 MBq of 99mTc pertechnetate in 400 to 500 μl saline to [99mTc(CO)3]+. After heating to 97°C for 30 min, the kit was neutralised with 1 M HCl and conversion to [99mTc(CO)3]+ was verified by thin-layer chromatography (TLC; glass-backed silica gel 60, Merck, Darmstadt, Germany; mobile phase: 1% HCl in methanol). The diabody was incubated at 37°C with 5.5 MBq/μg for 1 h and passed through a G25 Minitrap column (GE-Healthcare, Little Chalfont, UK) to remove residual unbound 99mTc and potential colloids. Labelling and final radiochemical purity were monitored by TLC in 60 mM citrate buffer, pH 5.5 (iTLCSA, Rf protein = 0, Rf [99mTc(CO)3]+ = 1) and analytical HPLC SEC (BioSep SEC-s2000, Phenomenex). Protein concentration after gel filtration was determined by UV absorption after decay using an aliquot stored at −80°C.
G25 minitrap column
Manufactured by GE Healthcare
Sourced in United Kingdom
The G25 Minitrap column is a lab equipment product from GE Healthcare. It is designed for chromatography applications, with a compact size and high performance. The core function of the G25 Minitrap column is to facilitate efficient separation and purification of various molecules and compounds.
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2 protocols using g25 minitrap column
1
Radiolabeling of Diabody Proteins
2
Fluorescent Labeling of BLS Proteins
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BLSDR-C and BLSKE-C were labeled with (i) Alexa Fluor 488C5 Maleimide and Alexa Fluor 555C5 Maleimide (Thermo Fisher Scientific), and (ii) Cy3 Maleimide and Cy5 Maleimide Mono-Reactive Dye (GE Healthcare), following the manufacturers' instructions. Briefly, proteins at 100 μM concentration in a Tris–HCl 50 mM buffer pH 7.3, supplemented with 1 mM of dithiothreitol (DTT) or 0.7 mM Tris(2-carboxyethyl)phosphine (TCEP), were incubated overnight at 4 °C in the presence of 2 M of the maleimide fluorophores. The reactions were stopped with the addition of β-mercaptoethanol. Then, the unbound dyes were removed using a G25 minitrap column (GE Healthcare). Finally, the labeled proteins were concentrated to ∼50 mg ml−1 stocks and stored at −80 °C for further use. The resulting labeled proteins were named A488BLSDR, A555BLSKE, Cy3BLSDR and Cy5BLSKE, respectively.
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