Concentration Determination Sample Clauses

Concentration Determination. To determine the concentration of protein on the AuNP, 32 L of conjugates were dissolved in a 16 L of a saturated potassium cyanide (KCN) solution. Once dissolved, 52 L of 10 mM sodium phosphate buffer was added for a total volume of 100 L. A standard curve was created using WT-DHFR such that 0.5 X, 0 X, 0 X, 0 M, 5 M, and 10 M were prepared in a similar manner as the conjugates, except 32 L of the free protein was used instead of conjugates. A fluorescence spectra for each sample was taken on Dual-FL fluorometer equipped with a Model 350B Temperature Controller set at 20C (Figure 4.1a). A standard curve of the WT standards was made from integrating the florescence spectra from 300 nm to 385 nm (Figure 4.1b). Once the curve was made, the conjugate samples were integrated, and the protein concentrations were found using the equation from the standard curve. This process was repeated with every set of conjugates to ensure proper concentration determination.

Concentration Determination. The concentration determination assay was able to adequately determine concentration of DHFR mutants bound to AuNPs. A novel concentration determination assay had to be developed Figure 4.6) because UV/Vis is could not determine concentration at such low concentration with scattering from AuNPs at 280 nm, and Bradford assays could not be performed due to the KCN causing the Xxxxxxxx xxx to degrade. Over many preparations, the range of the concentrations of the enzymes bound to AuNPs were consistent. The assay determined consistently between 2-5 M of protein bound to the 5 nm AuNPs in multiple preparations. For 15 nm AuNP conjugates, there was 2-4 M of protein bound, and there was approximately 2-5 M of protein bound to the 30 nm AuNP conjugates.