Nanodroptm one microvolume uv vis spectrophotometer

Circular dichroism far-UV and thermal denaturation measurements were recorded using a Jasco J-810 spectropolarimeter equipped with a CDF-426S Peltier temperature control unit (Jasco Products, OK, USA). The wild-type and mutant MBP–CHIP proteins were prepared at 2.5–5.8 µM concentration in a buffer containing 10 mM potassium phosphate and 100 mM sodium fluoride at pH 7.4. The protein concentrations were determined using a NanoDropTM One Microvolume UV–Vis spectrophotometer (Thermo Scientific, MA, USA), with theoretical extinction coefficients ε₂₈₀ = 96,720 (wild-type MBP–CHIP, G249V, and R51_I53delinsPA) and 91,220 (K143_W147del) M⁻¹cm⁻¹. Far-UV spectra were acquired in the range of 185–260 nm at a scan rate of 50 nm/min at 20 °C, using a quartz cell with a path length of 1 mm. Three scans were accumulated for each spectrum and three spectra were buffer subtracted and then averaged. Thermal denaturation profiles were obtained by recording the decrease in ellipticity at 222 nm as a function of temperature in the range of 20–90 °C with a scan rate of 40 °C/h. The results are expressed in mean residue ellipticity [θ]_mrw = θ(deg × cm² × dmol⁻¹), and BeSTSel (Beta Structure Selection) was used to estimate the secondary structure content [30 (link)]. Final graphs were prepared by using GraphPad Prism software (San Diego, CA, USA).

Following LPS stimulation, cells were collected by centrifugation at 300× g for 5 min, and total RNA was isolated from cells using an MG Total RNA Extraction Kit (MG Med, Seoul, Republic of Korea) according to the manufacturer’s instructions. The RNA purity and concentration were measured using a NanoDropTM One Microvolume UV-Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The transcription of pro-inflammatory genes, interleukin-6 (Il-6), tumor necrosis factor-α (Tnf-α), cyclooxygenase-2 (Cox-2), and inducible nitric oxide synthase (inos) was assessed by qRT-PCR using an MG One-Step RT-PCR MasterMix (SYBR Green, MG Med) and CFX ConnectTM Real-Time PCR detection system (Bio-Rad, Hercules, CA, USA). The following thermal cycling conditions were applied: 50 °C for 30 min, then 95 °C for 10 min, and then 40 cycles of 95 °C for 5 s and 60 °C for 40 s. The primers used for qRT-PCR are listed in Table 1. Eukaryotic translation elongation factor 2 (Eef2) was used as an internal reference gene. The relative transcription levels of pro-inflammatory genes were calculated by using the delta-delta Ct method (2−∆∆Ct) as previously described [13 (link)].

The peptides were solubilized in HEPES buffer and their concentration was determined using the Thermo Scientific^TM NanoDrop^TM One Microvolume UV–vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA, EUA), based on the absorbance of peptide’s backbone at 205 nm. Peptide concentration was calculated using the Scopes method from the NanoDrop One Protein A205 application, using the absorbance ratio A280/A205, to account for Tyr and Trp side chain absorbance. The linear dependence of absorption and fluorescence intensity over a peptide concentration range of 0–90 µmol dm⁻³ was studied as described by Ferre et al. [13 (link)]. The fluorescence properties of BP100 were evaluated at an excitation wavelength of 275 nm, in the wavelength range from 285–400 nm, using excitation/emission slit widths of 2.5 nm, while W-BP100 was excited at 280 nm, in the wavelength range from 300–450 nm, using excitation/emission slit widths of 2.0 nm.