New strategies to develop antibiotics for treatment of Pseudomonas aeruginosa and Leishmania spp infections.

All steps for the estimation of protein concentration were carried out using the Bradford dye-binding protein assay. This method is a colorimetric assay based on an absorbance shift of the dye-binding of Coomassie Brilliant Blue G250 to protein.
The materials used to measure the concentration of the E1 protein in PBS solution with this spectroscopic analytical procedure were:
1) the Coomassie Brilliant Blue G250 dissolved in phosphoric acid and methanol purchased from Sigma, as the assay reagent;
2) the protein standard BSA (bovine γ-globulin or bovine serum albumin) at a known concentration of 2 mg/ml in PBS (phosphate-buffered saline);
3) clean and detergent free disposable plastic cuvettes;
4) Eppendorf automatic pipettes and Eppendorf tubes.

The unknown protein concentration of the E1, which is a Fur P. aeruginosa like protein, was measured using a series of samples in Eppendorf tubes with increasing amounts of the protein standard as follows:
1) 300 µL Blue Bradford Reagent + 700 µL dH2O;
2) 0.1 µg/ml BSA;
3) 0.25 µg/ml BSA;
4) 0.5 µg/ml BSA;
5) 1.0 µg/ml BSA;
6) 2.0 µg/ml BSA;
7) 5.0 µg/ml BSA made up with PBS (total volume 50 µL)

At last were added 300 µL of Blue Bradford Reagent and 690 µL of distilled water to samples No.2 – No.7. Then, the samples were mixed and 1 ml of each one was transferred into the disposable plastic cuvettes provided. The No.1 was taken as the Blank, against which the absorbance at 595 nm of all the other samples was read in the spectrophotometer. So, a graph paper containing all these data (in Table 2) was made and also the standard curve related.
Finally, the unknown E1 protein sample was made up taking 10 µL of the protein PBS solution, to which were added 300 µL of Blue Bradford Reagent and 700 µL of distilled water (total volume 50 µL) and the absorbance was read in a spectrophotometer at 595 nm. Consequently, the E1 protein concentration was determined using this stan