Fig. 1. Purification of Tat-GFP by Q Sepharose Fast Flow chromatography. Bacterial lysate (200 ml) was loaded onto a QFF chromatography column. A step gradient of 0–100% Buffer B (250 ml) was applied (dashed line) and fractions were collected. A280 (thick line) was also continuously monitored. The recombinant Tat-GFP fusion protein was eluted as the first sharp single peak. Most proteins of the bacterial lysate did not bind to the resin.

Fig. 2. Western blot analysis of the monoclonal antibodies against GFP. The mAbs FMU-GFP.3 and FMU-GFP.5 displayed positive reaction to recombinant GFP at the concentration of 2 μg/ml. No reactivity was observed with normal mouse Ig. Numbers on left = molecular weight markers.

Fig. 3. Immunohistochemical analysis of anti-GFP mAbs. The mAb FMU-GFP.5 showed strong positive reaction with pEGFP-CD226 vector transfected CHO cells. (A) pEGFP-CD226 vector transfected CHO cells showed green fluorescence on the cell membrane; (B) mAb FMU-GFP.5 and PE conjugated goat-anti-mouse poAb (polyclonal antibody) staining; (C) dual-stain image (A and B). (For interpretation of color mentioned in this figure the reader is referred to the web version of the article.)

Fig. 4. Immunoprecipitation analysis of mAb against GFP. The mAbs FMU-GFP.3 and FMU-GFP.5 immunoprecipitated rGFP from E. coli lysate. Numbers on the left = molecular weight markers.

Fig. 5. SDS–PAGE analysis of purification of recombinant GFP fusion proteins by ion-exchange chromatography and mAb affinity column chromatography. Numbers on the left = molecular weight markers.

Fig. 6. FACS analysis of GFP protein transduction in CHO cell treated with affinity chromatography-purified tat-GFP. FACS analysis of CHO cells treated with Tat-GFP recombinant protein (0, 10, and 30 μg/ml, 1 h) demonstrated the Tat-GFP protein transduction efficiency was concentration dependent.

Fig. 7. Observation of GFP protein transduction in CHO cells treated with affinity chromatography-purified tat-GFP. CHO cells were treated with Tat-GFP recombinant protein (90 μg/ml, 30 min) demonstrated the Tat-GFP protein can enter cells efficiently.

Characterization of monoclonal antibodies against GFP