To estimate the cytosolic localization of antibodies, an enhanced split-GFP complementation assay was performed as previously described (18, 26). RT11-i-GFP11-SBP2, inCT37-GFP11-SBP2, inRas37-GFP11-SBP2, and inRas37-AAA-GFP11-SBP2 antibodies were prepared by coexpression of the respective GFP11 (residues 215 to 230)–SBP2 [streptavidin (SA)–binding peptide 2]–fused HC and its cognate LC in HEK293F cells, as described above. HeLa and SW480 cells stably expressing SA-fused GFP1 to GFP10 (residues 1 to 214) were generated as described before (18, 26) and designated as HeLa-SA-GFP1–GFP10 and SW480-SA-GFP1–GFP10 cells, respectively. These cells (2 × 104 cells per well), grown in black 96-well culture plates (SPL Life Sciences, 30296), were incubated at 37°C for 6 hours with one of the antibodies (0.1, 0.5, or 1 μM) in 50 μl of the medium. After washing with phosphate-buffered saline (PBS) to get rid of the autofluorescence of phenol red from the culture media, 50 μl of PBS was added into each well. The complemented GFP signals from cells were measured on a Cytation 3 multimode reader (BioTek, USA). The intact GFP was purified from E. coli, as described before (26). To construct a GFP fluorescence standard plot with known amounts of intact GFP, various amounts (0 to 0.5 ng) of the purified intact GFP were diluted twofold and added into wells of the same 96-well plate in 50 μl of PBS. GFP fluorescence intensity was read at 528 nm after excitation at 485 nm. The cytosolic amount and cytosolic concentration of iMabs were estimated via the formula described in detail elsewhere (26).

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.