PEI concentration Assay Bradford
Stock solutions of BSA, poly(Lys), poly(Arg), poly(Pro), poly(His), DGL (generations 1, 2, 3, 4, 5), PEI and PAMAM dendrimers were preparedat 100 µg mL−1 in ultrapure water purified from a MilliQ UV Plus Millipore system. Standard solutions were all freshly prepared from the above stock solutions avoiding successive dilutions. Water mixtures of the poly(amino acid)s or others macromolecules were at a final concentration of 0.2 to 40 µg mL−1 when processing the microplate modified Bradford assay as explained in the following section.
Microplate procedure Assays were carried out in 96 native microplate format wells as described by the supplier. The dye reagent was added to the stock macromolecule solution containing protein, linear or dendritic structures so that the dye concentration (DT) was maintained constant in 300 µL final volume per well with 1 : 1 CBB solvent : water mixture volume. After mixing the solution and waiting for 5 min in order to allow maximum dye binding, the absorbance was monitored at 620 nm. All experiments were repeated at least three times per sample concentration. When adding a macromolecule mixture to the dye solution, the observed change in absorbance (A) is then given by the following equation based on the Lambert–Beer law: A = (aBound − aFree)lDBound (1) where aBound and aFree are species molar absorptivities for bound and free dye under the microplate assay conditions with pH being about 1, DBound is the concentration of bound dye and l is the optical length path.
To optimize binding of selected macromolecule sites with dye, we carried out experiments at various dye/macromolecule ratios. We prepared a modified Bradford reagent following a procedure reported by Peterson.10 Briefly, CBB (20.5 mg) was dissolved in 95% ethanol (10 mL) followed by the addition of 85% phosphoric acid (20 mL). This mixture was completed with ultrapure water to 120 mL and sonicated. Solutions containing dye concentrations between 2 × 10−5 and 2 × 10−4 mol L−1 were prepared from the modified reagent. Free anion dye molar absorptivity (aFree) was estimated from a Beer’s law plot considering an optical path length l of 1.05 cm (well volume solution high). The aFree value was 14 199 L mol−1 cm−1. The obtained data were processed using a simplified Atherton mathematical model considering that only CBB blue species are taken into account during binding so that aCBB blue species = aBound. 5 A double reciprocal plot representation was drawn to provide a value of the absorptivity factor (a) and molar absorptivities of macromolecule-bound dye (aBound). Molar absorptivities were calculated under excess macromolecule conditions.