Overexpression of the Fab antibody was achieved successfully under the optimized condition (Figure 9(a)). described as Antibody Phage Display Methods and Protocols . The recombinants with light chain and pGEM-T were firstly digested by and (Fermentas, USA), then ligated into the Fd-pComb3 library, and transfected into XL1-Blue cells (Stratagene, CA, USA) repeatedly to obtain the Fab library ( 106?CFU). The presence and size of the inserts were further confirmed sequentially with and XL1-Blue cells in an exponential state and amplified as described above. Rescued phage particles were used to start a new selection round in ML-385 the Raf-1 same conditions according to the same protocol as described above . The phagemid titer was checked by counting the colony-forming unit (CFU) of the phagemid infected by XL1-Blue cells just before and after each panning. After the fifth round of screening, a total of 10 colonies were picked randomly and verified by a double digestion using enzymes and XL1-Blue was served as a black control. The absorbance was measured at 405?nm with a microplate reader (Model 550, Bio-Rad, CA, USA). Meanwhile, the optimal clone was further verified by the analysis of Western blot. Mouse anti-His antibody (1?:?2000) was used as a positive control, and crude cell extract of pComb3 clone was served as a negative control. Horse anti-mouse antibodies conjugated to alkaline phosphatase as the secondary antibody (1?:?2000) and BCIP/NBT (Amresco, CA, USA) as a chromogenic agent. Data obtained from the Western blot were analyzed by Bio-Rad Quantity One 1D Analysis software version 1.1 (Bio-Rad, CA, USA). Plasmid DNA from the optimal clone was purified and the inserts were completely sequenced and the deduced amino acid sequences were compared with DNA databank data using the BLAST program (National Center for Biotechnology Information, USA) to ascertain its sources. 2.6. Production of Soluble Fab Fragments The recombinant plasmid DNA from the clone number 29 was digested with and (MBI Fermentas, USA) for 2?h at 37C to remove the gIII fragment from pComb3, purified by using gel electrophoresis, and then self-ligated to build constructs for expression of soluble recombinant Fab. After the recombinant was identified by digestion, the clone was suspended in LB medium containing 100?cells were harvested by centrifugation at 2218?g for 15?min at 4C, and the pellet was suspended ML-385 with 20?mL of PBS and sonicated on ice. Crude cell extract with Fab fragments was obtained by centrifugation at 8,873?g for 30?min at 4C. 2.7. Purification of Fab The supernatant containing Fab prepared above was filtered by 0.22?mm filter membrane. The filtered solution was loaded onto Capto-L agarose chromatography column (HiTrap Protein L, GE) with the flow velocity of 1 1?mL/min. After washing out the unbound protein, the Fab was eluted out with sodium acetate buffer (pH 2.3) and neutralized with Tris-HCl (pH 8.0) immediately. Both flow-through unbound protein and eluted protein were collected for further verification by SDS-PAGE. 2.8. Analysis of the Characters of the Anti-P-gp Fab Fragment Expressed in XL1-Blue After purification, Fab concentration was determined using a BCA protein assay kit (Pierce Biotechnology, USA). The specificity of the purified Fab to ML-385 P-gp21 was also analyzed by Western blot using goat anti-mouse IgG conjugated to HRP (1?:?3000) (Southern Biotech, CA, USA). The moiety of BSA and the 15?kDa peptide expressed by BL21 (prepared by our lab) were served as the negative control. P-gp21 harboring three epitopes was chosen as the antigen according to its antigenicity estimated by using BepiPred 1.0b Server (Figure 1). Three peptides with strong antigenicity coupled to bovine serum albumin were synthesized (China Peptides Co. Ltd, Shanghai, China) for selection of the Fab. A 96-well microtiter plate (Nunc, Denmark) was coated with aliquot of BSA, 10-peptide-BSA, 12-peptide-BSA, and 16-peptide-BSA,.