Recombinant human wild-type ERp57 and the luminal domain of CNX were overexpressed in E. coli strain BL21 (DE3) and purified as described previously [37 (link),39 (link)]. Recombinant HLA-Cw4 heavy chain was overexpressed as inclusion bodies in the same cell type. Inclusion bodies were treated with 100 mM Tris/HCl (pH 8.0) buffer containing 8 M urea and 10 mM dithiothreitol. The proteins were purified by RP-HPLC (Hitachi High-Tech Corporation, Tokyo, Japan) using a Cosmosil 5C18-AR-II column (4.6 mm I.D. × 250 mm; Nacalai Tesque, Shiga, Japan) with monitoring at 220 nm and confirmation by mass spectrometry. The HLA-Cw4 heavy chain purified in the reduced/denatured state was lyophilized for storage.
Rp hplc
Manufactured by Hitachi
Sourced in Japan
The RP-HPLC (Reverse Phase High Performance Liquid Chromatography) is a laboratory instrument used for the separation, identification, and quantification of chemical compounds in a complex mixture. The core function of RP-HPLC is to separate, analyze, and measure the components of a sample based on their interactions with the stationary and mobile phases.
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Lab products found in correlation
Cosmosil 5c18 ar 2 column, by Nacalai Tesque (1 mentions)
Ika mag, by IKA Group (1 mentions)
Purospher star rp c18 column, by Merck Group (1 mentions)
400 spectrometer, by Bruker (1 mentions)
Finnigan mat95xl spectrometer, by Thermo Fisher Scientific (1 mentions)
Astm 9385 kieselgel 60h, by Merck Group (1 mentions)
Maldi tof ms, by Bruker (1 mentions)
7 protocols using rp hplc
1
Recombinant Expression and Purification of HLA-Cw4
2
Bufalin-BSA Nanoparticle Characterization and Release
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Particle morphology was investigated by TEM after negative stained with phosphonic acid wolfram. The average particle size, particle size distribution and Zeta potential of Bufalin-BSA-NP were determined by laser light scattering method. The as prepared Bufalin-BSA-NP emulsion was centrifugated (10,000 rpm/min, 4°C) for 30 min to acquire desalinized Bufalin-BSA-NP in the lower layer and free Bufalin solution in the up layer, of which the amount of Bufalin was measured by reverse-phase high-performance liquid chromatography (RP-HPLC, Hitachi Co., Tokyo) method. Encapsulation efficiency (EE%) and drug loading (DL%) were worked out by following equations: EE% = Bufalin in Bufalin-BSA-NP/total Bufalin × 100%; DL% = Bufalin in Bufalin-BSA-NP/weight of Bufalin-BSA-NP × 100%; where the weight of Bufalin-BSA-NP is equal to albumin plus Bufalin in Bufalin-BSA-NP.
Dialysis bag containing desalinized Bufalin-BSA-NP emulsion was put into 100 ml of phosphate buffer (pH 7.4). The buffer solution was oscillated in thermostatic oscillator (37 ± 1°C, 50 ± 1 shake frequency/min). At a specific time, 1 ml of suspension was taken out and the concentration of Bufalin was then measured with RP-HPLC. Finally, the accumulative release rate could be calculated with the concentration data measured.
Dialysis bag containing desalinized Bufalin-BSA-NP emulsion was put into 100 ml of phosphate buffer (pH 7.4). The buffer solution was oscillated in thermostatic oscillator (37 ± 1°C, 50 ± 1 shake frequency/min). At a specific time, 1 ml of suspension was taken out and the concentration of Bufalin was then measured with RP-HPLC. Finally, the accumulative release rate could be calculated with the concentration data measured.
3
Quantifying Detergent in Cassava Fufu
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The actual concentration of detergent absorbed in the detergent processed cassava (fufu) in each group was carried out using reversed-phase high performance liquid chromatography (RP-HPLC) (Hitachi; China). The final liquid chromatography analysis was performed on a Phenomenex Column (250 × 4 mm, 5 μm).
4
Quantifying Drug Release and Particle Stability
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After washing and drying, the powder was completely dissolved in 1 M HCl to determine the maximum release and loading efficiency. An aliquot was taken out and mixed with 700 µL of ethanol, 300 µL of 1 M HCl, and 50 µL of 0.02 M xanthyrol prior to the quantification with HPLC (RP-HPLC, Hitachi, Chiyoda, Japan).
Particle stability was determined by measuring drug release after incubating in phosphate buffer at pH 7.4. Additionally, 50 mg of particles were immersed in 10 mL of phosphate buffer and placed onto a shaker (IKAMAG®, IKA Werke GmbH & Co. KG, Staufen im Breisgau, Germany), at a speed of 50 rpm. An aliquot (300 µL) was taken out after 1 h, 1 day, 1 week, and 2 weeks.
Particle stability was determined by measuring drug release after incubating in phosphate buffer at pH 7.4. Additionally, 50 mg of particles were immersed in 10 mL of phosphate buffer and placed onto a shaker (IKAMAG®, IKA Werke GmbH & Co. KG, Staufen im Breisgau, Germany), at a speed of 50 rpm. An aliquot (300 µL) was taken out after 1 h, 1 day, 1 week, and 2 weeks.
5
Quantification of HU by RP-HPLC
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The quantification of HU was measured using reversed-phase high-performance liquid chromatography (RP-HPLC, Hitachi, Chiyoda, Japan) with UV detection (λ = 213 nm). Prior to injection in a column, the sample was mixed with 700 µL ethanol, 300 µL 1 M hydrochloric acid, and 50 µL 0.02 M xanthyrol in 1-propanol. HU is a small molecule that does not absorb UV and therefore, xanthyrol is added to act as a derivatization agent. An isocratic method was used with a mobile phase consisting of 50% 20 mM ammonium acetate (pH = 6.9) and 50% acetonitrile. The HPLC system was equipped with a Purospher® STAR RP-C18 column (150 mm × 4.6 mm, 5 μm, Merck, Darmstadt, Germany).
6
Spectroscopic Characterization of Synthesized Compounds
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Melting points were determined on a Mel-Temp melting point apparatus in open capillaries and were uncorrected. Infrared (IR) spectra were recorded using 1725XFT-IR spectrophotometer. High-resolution mass spectra (HRMS) were obtained on a Thermo Fisher Scientific Finnigan MAT95XL spectrometer using a magnetic sector analyzer. Peptide mass analysis was obtained by MALDI TOF MS (Bruker), and peptide purity was confirmed by RP-HPLC (Hitachi). 1H NMR (400 MHz) and 13C NMR (100) spectra were recorded on a Bruker 400 spectrometer. Chemical shifts were reported in parts per million on the scale relative to an internal standard (tetramethylsilane, or appropriate solvent peaks) with coupling constants given in hertz. 1H NMR multiplicity data are denoted by s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet). Analytical thin-layer chromatography (TLC) was carried out on Merck silica gel 60G-254 plates (25 mm) and developed with the solvents mentioned. Visualization was accomplished by using portable UV light, and an iodine chamber. Flash chromatography was performed in columns of various diameters with Merck silica gel (230–400 mesh ASTM 9385 Kieselgel 60H) by elution with the solvent systems. Solvents, unless otherwise specified, were reagent grade and distilled once before use. All new compounds exhibited satisfactory spectroscopic and analytical data.
7
Stability and Release of LXA4 Hydrogels
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To detect the stability of LXA4 in aqueous solutions, 100 µg/mL 15(R)-LXA4 in ethanol (Cayman Chemical) was diluted to 1 µg/mL using the 3 above aqueous buffers at different pH. After dilution, the samples (n = 3) were incubated at 37°C and the amount of LXA4 in the buffers at predetermined time points was detected by reverse-phase high-performance liquid chromatography (RP-HPLC; Hitachi) (for details, see Appendix ).
Release studies were performed at 37°C by a membraneless dissolution method (Zhang et al. 2002 (link)). Briefly, 200 µL of a PIC or P407 solution was mixed with 1 µg LXA4 on ice and then gelled in an Eppendorf at 37°C (n = 4). Subsequently, 1 mL PBS prewarmed at 37°C was laid over the gels. Samples were then incubated at 37°C with agitation at 200 rpm/min. At each predetermined time point, 900 µL supernatant was withdrawn carefully and 900 µL fresh PBS was refilled. The amount of released LXA4 in the supernatant was detected by RP-HPLC.
To further investigate the interactions between LXA4 and hydrogel carriers and influential factors of LXA4 release, the release study was also performed in media with detergent of 0.1% v/v Tween 20 in PBS (n = 4) and in buffers with different pH (n = 4): 1) PBS (pH 7.4), 2) HEPES (pH 8.5), and 3) citrate acid (pH 5) (Song et al. 2015 (link)).
Release studies were performed at 37°C by a membraneless dissolution method (Zhang et al. 2002 (link)). Briefly, 200 µL of a PIC or P407 solution was mixed with 1 µg LXA4 on ice and then gelled in an Eppendorf at 37°C (n = 4). Subsequently, 1 mL PBS prewarmed at 37°C was laid over the gels. Samples were then incubated at 37°C with agitation at 200 rpm/min. At each predetermined time point, 900 µL supernatant was withdrawn carefully and 900 µL fresh PBS was refilled. The amount of released LXA4 in the supernatant was detected by RP-HPLC.
To further investigate the interactions between LXA4 and hydrogel carriers and influential factors of LXA4 release, the release study was also performed in media with detergent of 0.1% v/v Tween 20 in PBS (n = 4) and in buffers with different pH (n = 4): 1) PBS (pH 7.4), 2) HEPES (pH 8.5), and 3) citrate acid (pH 5) (Song et al. 2015 (link)).
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