Mouse Anti-P. stutzeri TouR Recombinant Antibody (clone B7) (CAT#: FAMAB-0640WJ)

Figure 1 Binding of Phabs B7 and B9 to TouRΔA.

The binding of Phabs B7 and B9 and MBP (as a negative control ) to _6xhisTouRΔA or ovalbumin (OVA) as a specificity control antigen was determined by ELISA. Different dilutions of Phabs were incubated on ELISA plates coated with the antigens as indicated. After washing with PBS, the bound Phab was developed using anti-M13-peroxidase conjugate and the plates were read at OD₄₉₀. The data shown are relative to the maximal OD₄₉₀ obtained by Phab B7 at the higher titer employed (OD₄₉₀ of ∼2.0).

Fraile, S., Roncal, F., Fernández, L. A., & de Lorenzo, V. (2001). Monitoring intracellular levels of XylR in Pseudomonas putida with a single-chain antibody specific for aromatic-responsive enhancer-binding proteins. Journal of Bacteriology, 183(19), 5571-5579.

Figure 2 Recognition of XylR and TouR in Western blots by Phab B7.

Different protein preparations were separated by denaturing SDS-PAGE (10%), transferred to a PVDF membrane, and probed with the antibodies and Phabs indicated. (A) Different amounts of purified _6xhisTouRΔA (50, 16.6, 5.5, 1.8, and 0.61 ng [lanes 1 to 5, respectively]) were probed with a serum obtained from immunized mice (α-TouRΔA; top panel), Phab B7 (middle panel), or Phab MBP (as a negative control; bottom panel). (B) A mixture of 10 ng of purified _6xhisTouRΔA and _6xhisTouR (lane 1) or 30 ng of _6xhisXylRΔA and _6xhisXylR (lane 2) was probed with Phab B7. (C) Whole-cell protein extracts from P. stutzeri OX1 (lane 1) or P. putida KT2442 (lane 2) were probed with Phab B7. In all cases, the bound antibodies or Phabs were developed with anti-mouse-peroxidase or anti-M13-peroxidase conjugates, respectively.

Fraile, S., Roncal, F., Fernández, L. A., & de Lorenzo, V. (2001). Monitoring intracellular levels of XylR in Pseudomonas putida with a single-chain antibody specific for aromatic-responsive enhancer-binding proteins. Journal of Bacteriology, 183(19), 5571-5579.

Figure 3 Peptide mapping of the TouR epitope recognized by Phab B7.

(A) Summary of the binding of Phab B7 to three fragments derived from TouRΔA (F1, Leu226-Thr372; F2, Glu301-Val478; and F3, Leu439-Ala567). Recognition of these fragments by Phab B7 (+) was determined by Western blotting of whole-cell protein extracts of E. coli BL21(DE3) strains overproducing each fragment in pET vectors under the control of the T7 promoter (see Materials and Methods). (B) The amino acid sequence of TouR between positions Glu301 and Thr372 was synthesized onto cellulose membranes as deca- and dodecapeptides with an overlap of one amino acid. These membranes were probed with Phab B7 and developed with anti-M13-peroxidase conjugate. The results of these experiments are summarized, showing only the peptides around the TPRAQATLLR sequence, which forms the core epitope required for Phab B7 recognition. (C) The dodecapeptide TPRAQATLLRVL and a collection of derivatives in which each of the amino acid positions was changed consecutively to alanine (as indicated) were synthesized onto cellulose membranes and probed with Phab B7 as described for panel A. The change of any of the Arg residues (underlined) to Ala completely abolished Phab B7 recognition.

Fraile, S., Roncal, F., Fernández, L. A., & de Lorenzo, V. (2001). Monitoring intracellular levels of XylR in Pseudomonas putida with a single-chain antibody specific for aromatic-responsive enhancer-binding proteins. Journal of Bacteriology, 183(19), 5571-5579.