Figure 8Verification of Fab soluble expression vector by Not I digestion. (a) M: marker DL2000; lanes 1-2: number 1 and number 2 clones were digested by Spe I/Nhe I for deleting Fluoro-Sorafenib gIII fragment. (b) M: marker DL10000; lane 1: recombinant with no gIII deletion digested …Figure 9Fab expression and purification. (a) SDS-PAGE: 1, pComb3 clone was induced by 0.5mM IPTG at 28��C for 8h; 2, number 29 was not induced; 3, number 29 was induced by 0.5mM IPTG at 28��C for 6h; 4, number …3.8. Characterization of the Anti-P-gp Fab Fragment Expressed in E. coli XL1-BlueTo further confirm the purified Fab, P-gp21 was detected and estimated to be 21kDa in size when neither BSA nor 15-kDa peptide was used as the antigen by Western blot analysis (Figure 7(b)).

Three peptides with strong antigenicity, which belong to P-gp21 coupled to bovine serum albumin, were synthesized and used for identifying the exact epitope recognized by the Fab using an indirect immunofluorescence assay. Compared to either BSA, 10-peptide-BSA, or 12-peptide-BSA, the Fab showed high specificity bound to 16-peptide-BSA (Figure 10). These results were further confirmed by indirect immunofluorescence assay as shown in Figure 11.Figure 10Identification of the exact epitope recognized by the anti-P-gp Fab by indirect immunofluorescence.Figure 11Binding activity assay of anti-P-gp Fab to 16-peptide-BSA and BSA by using indirect immunofluorescence.An aliquot of BSA, 10-peptide-BSA, 12-peptide-BSA, and 16-peptide-BSA, at 1��g/��L, was coated on a 96-well microtiter plate. The anti-P-gp Fab (approximately 6��g/mL) was added and incubated at 37��C for 1h.

Fluorescein-isothiocyanate- (FITC-) conjugated goat anti-mouse IgG diluted 1:1000 is a secondary antibody for signal detection and crude cell extract of pComb3 cell was used as a negative control and secondary antibodies only as a background control. The relative fluorescence unit (RFU) was measured with an exitation spectrum at 490nm and emission spectrum at 520nm.Data for anti-P-gp Fab and cell extract of pComb3 cell were subjected to ANOVA; differences between means were determined using the least significant difference (LSD) statistic (P < 0.05).Microplates were coated with 16-peptide-BSA and BSA at 1��g/mL, respectively. Fab was titrated down by twofold dilution starting at 6��g/mL of antibody concentration. The second antibody was fluorescein-isothiocyanate- (FITC-) conjugated goat anti-mouse IgG. RFU was measured with an exitation spectrum at 490nm and emission Anacetrapib spectrum at 520nm.4. DiscussionIntrinsic or acquired resistance to a broad spectrum of chemotherapeutic agents often occurred in cancer patients who receive chemotherapy, resulting in failure of chemotherapy.