Recombinant Antibody to Green Fluorescent Protein (GFP) (RAD025Ge71)

Briefly, biotin labelled GFP Nano-antibody were diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100μl were then transferred to GFP-coated microtiter wells and incubated for 1h at 37℃. Wells were washed 3 times with PBST and incubated for 0.5 h at 37℃ with SA labelled HRP. After incubation with SA labelled HRP, wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50μL stop solution to the wells and read at 450/630 nm immediately. The binding activity of GFP and GFP Nano-antibody was shown in Figure 1, and the ED50 for this effect is 0.0187 ug/ml.

GFP was immunoprecipitated 293F whole cell extract (Expressing GFP) with 2 μg of RAD025Ge71 and 20 μl of Nickel pillar magnetic beads ( ). Western blot was perfomed on the immunoprecipitate using 0.05μg/ml MAD025Ge21 and 0.2μg/mL SAA544Mu19. Lane1 (input): 293F whole cell lysate. Lane2 ( ): RAD025Ge71 in 293F whole cell lysate. Lane3 (-): No antibody was added to the 293F whole cell lysate.