The availability of a recombinant anti-SNAP antibody in VHH format amplifies the application flexibility of SNAP-tagged proteins.
Antibodies are indispensable reagents in basic research, and those raised against tags constitute a useful tool for the evaluation of the biochemistry and biology of novel proteins. In this paper, we describe the isolation and characterization of a single-domain recombinant antibody (VHH) specific for the SNAP-tag, using Twist2 as a test-protein. The antibody was efficient in western blot, immunoprecipitation, immunopurification, and immunofluorescence. The sequence corresponding to the anti-SNAP has been subcloned for large-scale expression in vectors that allow its fusion to either a 6xHis-tag or the Fc domain of rabbit IgG2 taking advantage of a new plasmid that was specifically designed for VHH antibodies. The two different fusion antibodies were compared in immunopurification and immunofluorescence experiments, and the recombinant protein SNAP-Twist2 was accurately identified by the anti-SNAP Fc-VHH construct in the nuclear/nucleolar subcellular compartment. Furthermore, such localization was confirmed by direct Twist2 identification by means of anti-Twisit2 VHH antibodies recovered after panning of the same naïve phage display library used to isolate the anti-SNAP binders. Our successful localization of Twist2 protein using the SNAP-tag-based approach and the anti-Twist2-specific recombinant single-domain antibodies opens new research possibilities in this field.
Aliprandi
Marisa
M
Protein Chemistry Unit, Cogentech, IFOM-IEO Campus for Oncogenomics, Via Adamello 16, 20139 Milan, Italy.
Sparacio
Eleonora
E
Pivetta
Flavia
F
Ossolengo
Giuseppe
G
Maestro
Roberta
R
de Marco
Ario
A
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western; D004926:Escherichia coli; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000235:genetics*; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D019151:Peptide Library; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D013194:Staining and Labeling; D051879:Twist-Related Protein 1 / Q000276:immunology
D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D051879:Twist-Related Protein 1; D019853:O(6)-Methylguanine-DNA Methyltransferase