Du 730 life sciences uv vis spectrophotometer

The optical absorption spectra of the nanoparticles in water were evaluated using a Beckman Coulter DU^® 730 Life Sciences UV-Vis spectrophotometer. The fluorescence spectra were acquired using a TECAN M200 Infinite plate reader. The relative fluorescence quantum yield (ϕ) of the nanoparticles was calculated by comparing the ϕ of the standards Rhodamine 6G in ethanol (ƞ = 1.36, ϕ = 0.95) and Fluorescein in 0.1M sodium hydroxide (ƞ = 1.36, ϕ = 0.92) [56 ]. Quantum yields of the nanoparticles in water were calculated according to the equation below.
The slope of the line acquired from the plot of integrated fluorescence intensity versus absorbance is represented by (m) and ƞ represents the refractive index of the solvent used [56 ]. The R subscript denotes the reference fluorophore with a known quantum yield. The fluorescence spectra were measured by exciting the samples at 465 nm, collecting readings from 490 to 850 nm, baseline corrected, integrated, and plotted using SigmaPlot^® Software. The 465 nm excitation wavelength was chosen in order to align with the excitation parameters of our institution’s In Vivo Imaging System (IVIS).

Oligonucleotide primers were synthesized by Integrated DNA Technologies or Sigma-Aldrich. Recombinant plasmid DNA was purified with a QIAprep^® Spin Miniprep Kit from Qiagen. Gel extraction of DNA fragments and restriction endonuclease clean up were performed using an Illustra GFX PCR DNA and Gel Band Purification Kit from GE Healthcare. DNA sequencing was performed by Beckman Coulter Genomics and Eton Bioscience. Optical densities of E. coli cultures were determined with a DU 730 Life Sciences UV/Vis spectrophotometer (Beckman Coulter) by measuring absorbance at 600 nm. All chemicals and solvents were obtained from Sigma-Aldrich except where noted.
During protein purification, proteolysis was inhibited by adding Pierce™ Protease Inhibitor tablets (EDTA free; Thermo Scientific) to lysis buffer. Affinity chromatography and SDS-PAGE analysis were conducted using HisPur™ Nickel-nitrilotriacetic acid-agarose (Ni-NTA) resin (Thermo) and 10% or 4–15% Mini-PROTEAN TGX Precast SDS-PAGE gels (Bio-Rad) with 2× Laemmli sample buffer (Bio-Rad). Protein concentrations were determined by measuring absorbance at 280 nm with a NanoDrop 2000 spectrophotometer (Thermo).