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Understanding PadR Interactions: Western Blot and EMSA Analysis of p-Coumaric and o-Coumaric Acids

This study investigates the interactions of the PadR regulator with the padC promoter, revealing critical insights into its binding affinities for p-coumaric and o-coumaric acids in the presence of varying MgCl2 concentrations. We employed Western blot techniques to examine purified polyclonal PadR antibodies against crude protein extracts from recombinant E. coli strains. The results indicate that p-coumaric acid binding is significantly inhibited at magnesium concentrations below 0.25mM, while o-coumaric acid shows no significant effect. These findings provide important knowledge for future research on bacterial regulatory mechanisms.

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Understanding PadR Interactions: Western Blot and EMSA Analysis of p-Coumaric and o-Coumaric Acids

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  1. Tran et al, supplemental material P-cou 2 mM O-cou 2 mM pTZ pTR pTC BS P MgCl2 2.5 2.5 1 0.5 0,25 0 0.5 0,25 0 PadR probe OD600 = 1,6 Fig. S1. Western-blot of purified polyclonal PadR antibodies on crude protein extracts from recombinant E. coli strains. Antibodies were produced and purified by Eurogentec, Belgium, from rabbits immunized with PadR purified from recombinant E. coli strain BL21pER. pTZ, control strain with the no-insert pTZ19R vector; pTR, pTZ19R harboring and expressing the padR gene; pTC, pTZ19 expressing padC; and BS, crude extract from the B. subtilis wild-type strain (see Table 1 and Fig. 4). Crude extracts were resolved by SDS-PAGE and proteins were electro-transferred onto a nitrocellulose membrane prior to Western-blotting. Fig. S2. EMSA with the padC promoter probe and 1nM of PadR at different concentrations of MgCl2 from 0 to 2.5mM in the presence of 2 mM of p-coumaric (P-cou) or o-coumaric (O-cou) acid. P, padC promoter probe without protein. Effective inhibition of p-coumaric acid binding was detectable when MgCl2 concentrations were lower than 0.25mM. No effect was detected with o-coumaric acid even in the absence of MgCl2 (For EMSA conditions, refer to the legend of Fig. 6).

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