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Figure 5: Effects of Ex-phage packaging on antigen-binding reactivity of scFv displayed on recombinant phage. (A) Determination of scFv:pIII fusion protein expression by immunoblot. Recombinant phage particles were obtained by infecting JS5 cells carrying pIGT3-hsp70 phagemid with either M13KO7 helper phage (pIGT3/ M13KO7) (lane 1) or Ex-phage (pIGT3/ Ex-phage) (lane 2). Phage proteins were separated by loading approximately 1010 recombinant phage into each lane of 10% SDS–PAGE, and immunoblot was carried out with anti-gIII mAb to determine the amount of scFv:pIII fusion proteins displayed on the surfaces of phage particles. Goat anti-mouse IgG antibody conjugated with HRPO was used for the secondary antibody. (B) Antigen-binding specificity of recombinant phage packaged with either M13KO7 or Ex-phage. BSA, lysozyme (Sigma Co.), recombinant GST or recombinant human HSP-70 were coated in microtiters, and 1010 scFv phage packaged with either M13KO7 or Ex-phage were applied to each well for phage ELISA. The same amount of M13KO7 helper phage was used as a negative control. The bound phage were detected with anti-M13 antibody conjugated with HRPO. The binding signal was analyzed at OD405. (C) Antigen-binding sensitivity of phage particles packaged with either M13KO7 or Ex-phage. Phage ELISA was performed as described above except that serial dilutions of purified human HSP-70 fusion protein were used to coat microtiter plates.
Image Text (High Precision): 0.1 405 A.D. Ex TG1 antigen concentrations fusion lysozyme m13k07 pIGT3 pIGT3/Ex pIII phage preparations scFv
Other Images from "An improved helper phage system for efficient isolation of specific antibody molecules in phage display":
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Figure 6 Enhancement of panning efficiency by Ex-...
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