The previously described anti-F1 scFv antibodies58 (link) were converted to IgGs by inserting the amino-acid sequences corresponding to the variable heavy (VH) and variable light (VL) antibody regions into a standard IgG1 scaffold. The resulting protein sequences were submitted to ATUM (Newark, CA, USA) for codon-optimized back-translation, gene synthesis, and expression as full-length IgG1 antibodies in HEK293 cells. Commercial anti-F1 YPF19 from Advanced ImmunoChemical (Long Beach, CA, USA) was used as positive control and natural human IgG1 (Abcam, Cambridge, MA, USA) as negative control in various binding assays. In addition, scFv and IgG formats of an anti-influenza M2 scFv59 (link) were also used as negative controls, since these antibodies use the same vector systems as the anti-F1 antibodies described here. Enzymatic and/or fluorescent labeling of antibodies (eg, horseradish peroxidase [HRP], phycoerythrin [PE], and allophycocyanine [APC]) was performed using Lightning Link kits (Novus Biologicals, Centennial, CO, USA) according to the manufacturer’s instructions. All antibody-binding assays were performed at 25°C.
Lightning link kits
Manufactured by Novus Biologicals
Sourced in United States
The Lightning Link kits are laboratory tools designed for rapid and convenient protein conjugation. They enable the simple and efficient coupling of proteins, antibodies, or other biomolecules to a variety of labels or detection reagents.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using lightning link kits
1
Conversion of anti-F1 scFv to IgG1
2
Immunofluorescence Staining of Cells
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Cells were washed twice with PBS and adhered to covers pre‐coated with poly‐l ‐lysine, followed by fixation with 4% paraformaldehyde for 10 min at room temperature. Cells were then washed twice with PBS and permeabilized with PBS containing 1% Triton X‐100 for 10 min, followed by washing with PBS for twice and blockage in 10% normal goat serum for 30 min at 37°C. Cells were incubated with primary antibody for 2 h, followed by washing with PBS for three times and further incubation with fluorescence‐conjugated secondary antibody for 1 h. Nuclei were stained with DAPI. For co‐immunostaining using antibodies with the same origin, Lightning‐Link kits (ab236553 and ab269900, Novus Biologicals) were used to directly link fluorescence to primary antibodies. Cells were visualized through an UltraVIEW VoX imaging system (PerkinElmer).
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