Monoclonal primary antibodies, NS5A (9E10; gift from Timothy Tellinghuisen), NS3 and NS5A (Virostat; 1877), were labelled with Alexa Fluor 647 NHS ester (Life Technologies) at an antibody/dye ratio of ~1:1 (see Supplementary Table 1 ). Fluorescent dye (0.1 µg) was incubated with 3 µl primary antibody (1 mg/ml) and 125 mM NaHCO3 in PBS for 30 min in the dark. Labelled antibodies were recovered from excess unreacted fluorescent dye using 40 K MWCO Zeba Spin Desalting columns (Thermo Fisher Scientific) following the manufacturer’s instructions. Fluorescence labelling of antibodies was confirmed by measuring the absorbance trace at 280 nm and 665 nm.
40 k mwco zeba spin desalting columns
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 40 K MWCO Zeba Spin Desalting columns are a lab equipment product designed for desalting and buffer exchange. The columns utilize a size-exclusion chromatography technique to remove small molecules, such as salts, from larger protein samples.
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3 protocols using 40 k mwco zeba spin desalting columns
1
Fluorescent Labeling of Monoclonal Antibodies
2
Fluorescent Labeling and Gel Shift Assay for dCas9
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The Halo domain of dCas9 was labeled by incubation with fluorescent Halo ligand (G1002 HaloTag® Alexa Fluor® 488 ligand, Promega) at ratio of 1:10 at room temperature for 30 min, and purified using 40-K MWCO Zeba spin desalting columns (Thermo Scientific). Gel mobility shift assay was carried out in 1 × EMSA buffer (38 (link)). dCas9 protein was preincubated for 10 min with 9 μl of EMSA buffer. Then 1 μL gRNA was added, and the reaction was incubated at room temperature for 15 min to examine the binary complex. Samples were then resolved by 6% native PAGE at 4°C (0.5 × TBE buffer). After electrophoresis (100 V, 1.0 h), in gel targets were visualized using a Pharos FX Molecular imager (Bio-Rad, USA) in the fluorescence mode (λex = 488 nm).
3
Site-Specific Protein Labeling with Fluorescent Dyes
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BG-modified or SNAP-Surface® Alexa Fluor® 647 (New Englands Biolabs, Ipswich, MA, USA) were conjugated to scFv-SANP fusion proteins by incubating at a 2:1 molar ratio for 2 h at room temperature in the dark. The residual agents were removed by 40K MWCO Zeba ™ Spin Desalting Columns (Thermo Fisher Scientific, Rockford, IL, USA). The site-specific conjugation was confirmed by post-incubation with SNAP-Surface® Alexa Fluor® 488. SNAP-Surface® Alexa Fluor® 488 and SNAP-Surface® Alexa Fluor® 647 fluorescent were visualized under UV light using ChemiDoc XRS+ System (BIO-RAD, Hercules, CA, USA) and Odyssey DLx Imager (LI-COR Biosciences, Bad Homburg, Germany) individually after protein separation in SDS-PAGE.
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