Large-winged pearl shells (Pteria penguin, 6 years old) were provided by Amami South Sea & Mabe Pearl Co. Ltd. (former Amami-branch, Tasaki & Co. Ltd.), Kagoshima, Japan. Mantle and its secretory fluid were collected and stored at −80 °C or −30 °C until use, respectively. A POROS® HS column was purchased from Applied Biosystems Thermo Fisher Scientific (Waltham, MA, USA). Resource S and HiTrap NHS-activated columns were purchased from GE Healthcare UK Ltd. (Amersham Place, Buckinghamshire, UK). TSKgel ODS-120T, COSMOSIL Protein-R, and CAPCELL PAK columns were purchased from Tosoh (Tokyo, Japan), SHISEIDO (Tokyo, Japan) and Nacalai tesque (Kyoto, Japan), respectively. Achromobacter protease I and Staphylococcus aureus V8 protease were purchased from Wako Pure Chemical Ind. (Osaka, Japan). Trehalose was purchased from Hayashibara (Okayama, Japan). PA-oligosaccharides were purchased from TaKaRa Bio Co. (Kyoto, Japan). All other reagents were of the purest grade commercially available.
Tskgel ods 120t
Manufactured by Tosoh
Sourced in Japan
The TSKgel ODS-120T is a reversed-phase high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of organic compounds. It features a porous silica-based stationary phase with octadecylsilane (ODS) bonding, providing a high-quality separation performance.
Automatically generated - may contain errors
Lab products found in correlation
Poros hs column, by Thermo Fisher Scientific (1 mentions)
1100 series, by Agilent Technologies (1 mentions)
Jes fr 30 free radical monitor, by JEOL (1 mentions)
Sepharose cl 4b, by Cytiva (1 mentions)
High performance liquid chromatography (hplc), by Shimadzu (1 mentions)
Resource s column, by GE Healthcare (1 mentions)
Sephadex g 15, by GE Healthcare (1 mentions)
Sepharose 4b, by GE Healthcare (1 mentions)
Staphylococcus aureus v8 protease, by Fujifilm (1 mentions)
Mucin from porcine stomach type 2, by Merck Group (1 mentions)
6 protocols using tskgel ods 120t
1
Isolation and Characterization of Pearls
2
HPLC Analysis of Compound Quantification
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The HPLC method adapted from the literature25 (link) used a TSKgel ODS-120T 150×4.6 mm, 5 µm particle column coupled with a TSKgel ODS-120T 3.2×1.5 cm (Tosoh Bioscience, Tokyo, Japan) guard gel. The column was kept at 37°C. The mobile phase was a mixture of Milli-Q water–TFA 0.1% v:v (phase A) and acetonitrile–TFA 0.1% v:v (phase B). The mobile-phase gradient used was linear, from 12% (0 minutes) to 52% (32 minutes) in phase B. The wavelength was fixed at 210 nm, and the flow rate was set at 1 mL/min. The lower limit of quantification was 5 µg/mL. The HPLC system used was a 1100 series from Agilent (Santa Clara, CA, USA).
3
HPLC-ESR Analysis of Radical Adducts
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An HPLC used in the HPLC-ESR consisted of a model 7125 injector (Reodyne, Cotati, CA), a model L-7100 pump (Hitachi Ltd., Ibaraki, Japan). A semi-preparative column (300 mm long × 10 mm i.d.) packed with TSKgel ODS-120T (TOSOH Co., Tokyo, Japan) was used. Flow rate was 2.0 ml/min throughout the HPLC-ESR experiments. For the HPLC-ESR, two solvents were used: solvent A, 50 mM acetic acid; solvent B, 50 mM acetic acid/acetonitrile (20/80, v/v). A following combination of isocratic and linear gradient was used: 0–40 min, 100% A to 20% A (linear gradient); 40–60 min, 80% B (isocratic). The eluent was introduced into a model JES-FR30 Free Radical Monitor (JEOL Ltd.). The ESR spectrometer was connected to the HPLC with a Teflon tube, which passed through the center of the ESR cavity. The operating conditions of the ESR spectrometer were: power, 4 mW; modulation width, 0.2 mT; time constant, 1 s. The magnetic field was fixed at the third peak in the double-triplet ESR spectrum (αN = 1.58 mT and αHβ = 0.26 mT) of the 4-POBN radical adducts (Fig. 1 ).
4
Liposomal Entrapment Efficiency Determination
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The encapsulation efficiency of liposomes was examined after separating free PMX from liposomes by Sepharose CL-4B (Amersham Bioscience, Uppsala, Sweden) column chromatography. 27) (link) Entrapped PMX was then determined by lysis of liposomes with methanol. The PMX content was analyzed by a high performance liquid chromatography (HPLC, Shimadzu, Kyoto, Japan) equipped with a C 18 column (TSKgel ODS120T, TOSOH Bioscience) of a 4.6 mm×150 mm size. Phosphate buffer-acetronitrile (80 : 20) was used as a mobile phase at flow rate of 1 mL/min, an injection volume of 5 µL and at a wavelength of 254 nm. The PMX concentration was determined from the calibration curve of PMX at various concentrations. The experiment was independently performed for 3 repeating samples per experimental group (n=3). Experimental and theoretical percentages of PMX loading were calculated from Eqs. 1 and 2, respectively:
5
Purified Enzyme Proteome Analysis
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Purified enzyme (100 μg) was separated on SDS-PAGE and visualized by CBB staining. The expected protein band was excised with a sharp blade and washed with water. The gel containing protein band was cut into pieces and destained with 40% acetonitrile in 100 mM ammonium carbonate for 24 h. Then protein in gel was reduced with DTT (10 mM) at 56 °C and alkylated with iodoacetamide (50 mM) in 100 mM ammonium carbonate for 1 h in every treatment. The polyacrylamide gel was dehydrated with acetonitrile after completion of each treatment. The reduced and alkylated proteins were digested with lysylendopeptidase at protein to peptidase ratio 50:1 in 50 mM Tris–HCl buffer, pH 8.5 for 24 h. After digestion, the peptides were extracted from polyacrylamide gel once with 1.0% formic acid, once with 9% acetonitrile in 20 mM ammonium carbonate and twice with 60% acetonitrile in 0.1% formic acid. The extracted peptides were lyophilized by freeze drying, reconstituted in water and subjected to RP-HPLC separation on a column TSK gel ODS-120 T (4.6 × 150 mm, Tosoh, Tokyo, Japan) at a flow rate 1 ml min−1 followed by elution with a 90 min linear gradient of acetonitrile (5-50%) in 0.1% trifluroacetic acid (TFA). The peptide peaks were detected at 215 nm.
6
Isolation and Characterization of Pearl Shell Proteins
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Cultured large winged pearl shells (Pteria penguin, 6-years-old) and their larva were kindly provided by Amami South Sea & Mabe pearl Co. Ltd. (former Amami-branch, Tasaki & Co. Ltd.), Kagoshima, Japan. The mantle was collected and stored at −80°C until use. Resource S column, Sephadex G-15 and Sepharose 4B gels were purchased from GE Healthcare (NJ, USA). TSKgel sugar AXI, TSKgel ODS 120T, and TSKgel Amide 80 columns were from Tosoh (Tokyo, Japan). Trehalose-Sepharose 4B gel was prepared according to the method of Matsumoto et al. [29] . Mucin type I from bovine submaxillary glands and mucin type II from porcine stomach were purchased from Sigma Chemical (St. Louis, MO, USA). Achromobacter protease I and Staphylococcus aureus V8 protease were purchased from Wako Pure Chemical (Osaka, Japan). Glycopeptidase A was purchased from Seikagaku Kogyo (Tokyo, Japan). Trehalose was purchased from Hayashibara (Okayama, Japan). All the other reagents were of the purest grade commercially available.
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