Editing Colorimetric quantification of amino groups

&nbsp;Colorimetric quantification of amino groups



Preparation of standard solutions<br>Stock solutions of BSA, poly(Lys), poly(Arg), poly(Pro), poly(His),<br>DGL (generations 1, 2, 3, 4, 5), PEI and PAMAM dendrimers were<br>prepared at 100 μg mL −1 in water (18 M ) purified from a MilliQ UV<br>Plus Millipore system. Standard solutions were all freshly prepared<br>from the above stock solutions avoiding successive dilutions.<br>Water mixtures of the poly(amino acid)s or others macromolecules<br>were at a final concentration of 0.2 to 40 μg mL −1 when processing<br>the microplate modified Bradford assay as explained in the<br>following section.



Microplate procedure<br>Assays were carried out in 96 native microplate format wells<br>as described by the supplier. The dye reagent was added to<br>the stock macromolecule solution containing protein, linear or<br>dendritic structures so that the dye concentration (D T ) was<br>maintained constant in 300 μL final volume per well with 1 : 1<br>CBB solvent : water mixture volume. After mixing the solution<br>and waiting for 5 min in order to allow maximum dye binding,<br>the absorbance was monitored at 620 nm. All experiments were<br>repeated at least three times per sample concentration. When<br>adding a macromolecule mixture to the dye solution, the observed<br>change in absorbance ( A) is then given by the following equation<br>based on the Lambert–Beer law:



deltaA = (alpha Bound − alpha Free )/D Bound

where alpha Bound and alpha Free are species molar absorptivities for bound<br>and free dye under the microplate assay conditions with pH being<br>about 1, D Bound is the concentration of bound dye and l is the<br>optical length path.



Determination of molar absorptivities<br>To optimize binding of selected macromolecule sites with dye,<br>we carried out experiments at various dye/macromolecule ratios.<br>We prepared a modified Bradford reagent following a procedure<br>reported by Peterson. 10 Briefly, CBB (20.5 mg) was dissolved in<br>95% ethanol (10 mL) followed by the addition of 85% phosphoric<br>acid (20 mL). This mixture was completed with ultrapure water to<br>120 mL and sonicated. Solutions containing dye concentrations<br>between 2 × 10 −5 and 2 × 10 −4 mol L −1 were prepared from<br>the modified reagent. Free anion dye molar absorptivity (a Free )<br>was estimated from a Beer’s law plot considering an optical path<br>length l of 1.05 cm (well volume solution high). The a Free value was<br>14 199 L mol −1 cm −1 . The obtained data were processed using<br>a simplified Atherton mathematical model considering that only<br>CBB blue species are taken into account during binding so that<br>a CBB blue species = a Bound . 5 A double reciprocal plot representation<br>was drawn to provide a value of the absorptivity factor ( a)<br>and molar absorptivities of macromolecule-bound dye (a Bound ).<br>Molar absorptivities were calculated under excess macromolecule<br>conditions





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