Du 730 nucleic acid protein analyzer

Total phenolic content in ramie leaves were determined using the Folin-Ciocalteu colorimetric method with modification [12] , [13] (link). In brief, standard curve using gallic acid (0.0–600.0 µg/mL) was made. For each analysis, 100 µL of the standard gallic acid solution or extracts sample were added to 0.4 mL of water in each test tube. Folin-Ciocalteu reagent (0.1 mL) was added to the solution and allowed to react for 6 min to ensure that the Folin-Ciocalteu reagent reacted completely with the oxidizable phenolates in the sample. Second, 1 mL of 7% sodium carbonate solution was added to neutralize the reaction, and 0.8 mL of deionized water was added into the test tubes to adjust the final volume to 2.4 mL. The samples were mixed and allowed to stand for 90 min at room temperature. The absorbance was measured at 760 nm after the color was developed by DU 730 Nucleic Acid/Protein Analyzer (BECKMAN, USA). The absorbance values were calculated based on the standard curve of known gallic acid concentrations and expressed as milligram of gallic acid equivalents (GAE) per 100 g fresh weight (FW). Data were reported as mean ± SD for triplicates.

Optical rotations were determined using an MCP200 automatic polarimeter (Anton Paar, Graz, Austria). Ultraviolet spectra were recorded on a DU 730 nucleic acid/protein analyzer (Beckman Coulter, Brea, CA, USA). IR spectra (film) were measured with a Tensor 27 FT-IR spectrometer (Bruker, Ettlingen, Germany). 1D and 2D NMR spectra were recorded on a Bruker AV-600 spectrometer, δ in ppm rel. to TMS, J in Hz. ESIMS were recorded using an 1290-6420 Triple Quadrupole LC-MS spectrometer (Agilent, Santa Clara, CA, USA). HRESIMS were recorded with an Agilent G6230 TOF mass spectrometer. Silica gel (100–200 mesh, 300–400 mesh, Qingdao Marine Chemical Ltd., Qingdao, China), Sephadex LH-20 (GE Healthcare Bio-sciences AB, Uppsala, Sweden), YMC*GEL ODS-A (S-50 μm, 12 nm) (YMC Co., Ltd., Kyoto, Japan), and reversed-phase HPLC (Rohm and Hass Shanghai Chemical Industry Co., Ltd., Shanghai, China) were used for column chromatography. Biological assays were analyzed using a microplate reader (BioTek Synergy H1, BioTek Instruments, Winooski, VT, USA).

The Folin-Ciocalteu colorimetric method, as described earlier [49 ], with modifications [50 (link)], was used to determine the total phenolic content in the peel and pulp samples. All extracts were diluted with Milli-Q water to get readings falling within the range of the standard curve concentration: 0.0–600.0 µg gallic acid/mL. One hundred microliters of gallic acid solution or extracts were added to 0.4 mL of Milli-Q water in each test tube, followed by the addition of Folin-Ciocalteu reagent (0.1 mL). The solutions were allowed to react for 6 min to ensure the complete and speedy reaction of the Folin-Ciocalteu reagent with oxidizable phenolates in the sample. Then, 1 mL of 7% sodium carbonate solution was added to neutralize the mixture, followed by the addition of 0.8 mL Milli-Q water to adjust the final volume to 2.4 mL. The samples were mixed and allowed to stand for 90 min at room temperature. After color development, absorbance was measured at 760 nm on a DU 730 Nucleic Acid/Protein analyzer (BECKMAN, Inc., Fullerton, CA, USA). Total phenolic contents were calculated based on the standard curve of known gallic acid concentrations, and final values were expressed as milligrams of gallic acid equivalent per 100 grams on a fresh weight basis (mg GAE/100 g FW). Data were presented as the mean ± SD for triplicates analyses.

Total cellular RNA was extracted from the tissue samples using an RNeasy Mini kit (Qiagen) according to the manufacturer’s instruction. Purified total RNA was assessed for purity by measuring the absorbance at 260 and 280 nm using the DU730 Nucleic Acid/Protein Analyzer (Beckman Coulter, Inc., Brea, CA, USA), and stored at −80°C prior to analysis. Total RNA was reverse-transcribed using a SuperScript^® III First Strand kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions.

Total histone was extracted from PBMCs using the EpiQuik™ total histone extraction kit (Cat. P-3017-96, Epigentek Group Inc., NY, USA) according to the manufacturer's instructions. Briefly, every 1 × 10⁶ cells were lysed with 10 μL lysis buffer, mixed with three volumes of extraction buffer/glycerol solution by vortexing, and incubated on ice for 5 min. After centrifugation at 12,000 rpm for 5 min at 4°C, the supernatant was collected into a 1.5-mL tube, mixed with 100% TCA solution to a final concentration of 25%, and incubated on ice for 30 min to precipitate proteins. The pellet was collected by centrifugation at 12,000 rpm for 10 min at 4°C, washed twice with acetone, and dissolved in 10 μL of water per amount of pellet extracted from 1 × 10⁶ cells. The protein concentration was determined using Beckman Coulter DU 730 Nucleic Acid/Protein Analyzer, and the extracted histone protein samples were aliquoted and stored at −80°C.