Reagents were of ACS grade or higher (e.g. HPLC and Optima LC/MS) and were purchased from Thermo Fisher Scientific and used without further purification. Testosterone, epiTestosterone (epi-T), DHEA, DHEA-S sodium salt, DHEA glucuronide (DHEA-G), AST, epi-AST, DHT, 5-adiol, 3α-adiol and 3β-adiol were purchased from Steraloids (Wilton, NH, USA). [2,3,4-13C3]-T ([13C3]-T) and [2,3,4-13C3]-DHT ([13C3]-DHT) were from C/D/N Isotopes (Point-Claire, Quebec, Canada) and Cambridge Isotopes (Andover, MA, USA), respectively. [2,3,4-13C3]-3α-adiol ([13C3]-3α-adiol) and [2,3,4-13C3]-3β-adiol ([13C3]-3β-adiol) were synthesized by an enzymatic method according to our published procedure (Zang et al. 2017 (link)). 4-Dimethylaminopyridine (DAP) 2-methyl-6-nitrobenzoic anhydride (MNBAn), picolinic acid (PA), triethylamine (TEA), anhydrous tetrahydrofuran (THF), β-glucuronidase from E. coli and sulfatase from Abalone entrails were from Sigma-Aldrich. Charcoal dextran stripped fetal bovine serum (CD-FBS) was from Atlanta Biologicals (Lawrenceville, GA, USA).
Sulfatase from abalone entrails
Manufactured by Merck Group
Sourced in Japan
Sulfatase from abalone entrails is a natural enzyme derived from the digestive system of abalone, a type of marine gastropod mollusk. The core function of this product is to catalyze the hydrolysis of sulfate esters, breaking down complex sulfated compounds. This enzyme can be utilized in various research and industrial applications that require the processing or analysis of sulfated biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
High performance liquid chromatography (hplc), by Thermo Fisher Scientific (1 mentions)
Testosterone, by Steraloids (1 mentions)
3α adiol, by Steraloids (1 mentions)
3β adiol, by Steraloids (1 mentions)
β glucuronidase from e coli, by Merck Group (1 mentions)
Charcoal dextran stripped fetal bovine serum cd fbs, by Atlanta Biologicals (1 mentions)
Optima lc ms, by Thermo Fisher Scientific (1 mentions)
β glucuronidase from helix pomatia, by Merck Group (1 mentions)
Protein deglycosylation mix 2, by New England Biolabs (1 mentions)
3 protocols using sulfatase from abalone entrails
1
Steroid hormone quantification protocol
2
Enzymatic Hydrolysis of EGCG
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Standards of phenol, PC, and p-chlorophenol were purchased from Fujifilm Wako Pure Chemical Co.
(Osaka, Japan). Both β-glucuronidase from Helix
pomatia and sulfatase from abalone entrails were purchased
from Sigma-Aldrich Japan K.K. (Tokyo, Japan). A commercial EGCG product
(>94% of purity) was obtained from DSM Nutrition Japan, K.K. (Tokyo,
Japan). EGCG hydrolysate (a mixture of EGC and GA) was prepared by
enzymatic hydrolysis of EGCG according to a previous procedure with
a slight modification.26 (link) One gram of enzyme
preparation (tannase-KTFHR, Kikkoman Co., Chiba, Japan), which consisted
of 0.9% (w/w) tannase from Aspergillus oryzae, 99.0% glucose, and 0.1% inositol, was added to an aqueous solution
of EGCG (10 g/L) and incubated at 37 °C for 60 min. The reaction
mixture was evaporated and freeze-dried. The disappearance of EGCG
after tannase treatment was confirmed by high-performance liquid chromatography
(HPLC) (Figure S1 ). Except for EGC and
GA, newly generated peaks were undetectable.
(Osaka, Japan). Both β-glucuronidase from Helix
pomatia and sulfatase from abalone entrails were purchased
from Sigma-Aldrich Japan K.K. (Tokyo, Japan). A commercial EGCG product
(>94% of purity) was obtained from DSM Nutrition Japan, K.K. (Tokyo,
Japan). EGCG hydrolysate (a mixture of EGC and GA) was prepared by
enzymatic hydrolysis of EGCG according to a previous procedure with
a slight modification.26 (link) One gram of enzyme
preparation (tannase-KTFHR, Kikkoman Co., Chiba, Japan), which consisted
of 0.9% (w/w) tannase from Aspergillus oryzae, 99.0% glucose, and 0.1% inositol, was added to an aqueous solution
of EGCG (10 g/L) and incubated at 37 °C for 60 min. The reaction
mixture was evaporated and freeze-dried. The disappearance of EGCG
after tannase treatment was confirmed by high-performance liquid chromatography
(HPLC) (
GA, newly generated peaks were undetectable.
3
Deglycosylation and Sulfatase Treatment of Vaccines
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To deglycosylate the vaccine, 25 μg of the 2020 QIV was denatured for 10 min at 75°C and treated with protein deglycosylation mix II (New England Biolabs) for 30 min at 25°C and 1 h at 37°C. For sulfatase treatment, A/Hong Kong/485197/2014 H3N2 virus was diluted to 160 HAU in sodium acetate (pH 5.0) and treated with 20 units/ml of sulfatase from abalone entrails (Sigma-Aldrich) for 1 h at 37°C. As a control, equal quantities were diluted and incubated but did not receive the deglycosylation mix II or sulfatase enzymes and are referred to as untreated. After treatment, preparations underwent buffer exchange with PBS to remove freed glycans and sulfate groups. ELISA plates were coated at 1 μg/ml for the 2020 QIV or 8 HAU for the virus.
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