P. falciparum cultures were prepared as described previously (21 (link), 29 (link)). Erythrocytes were then lysed with 0.0125% saponin (Sigma, sapogenin content ≥ 10%) in cold phosphate-buffered saline (PBS) with an EDTA-free protease inhibitor mixture (Roche or Pierce). The released P. falciparum parasites were then washed in cold PBS with EDTA-free protease inhibitor mixture. The washed cell pellets were flash frozen in liquid nitrogen and stored at −80 °C.
Sapogenin
Manufactured by Merck Group
Sourced in United States
Sapogenin is a naturally occurring compound derived from plant sources. It serves as a key precursor in the synthesis of various steroid-based pharmaceuticals and chemical compounds. Sapogenin exhibits complex molecular structures and possesses important biochemical properties that make it a valuable raw material for the pharmaceutical and chemical industries.
Automatically generated - may contain errors
Lab products found in correlation
Saponin, by Merck Group (3 mentions)
Quillaja saponin, by Merck Group (1 mentions)
7 dehydrocholesterol, by Merck Group (1 mentions)
β sitosterol, by Merck Group (1 mentions)
2 hydroxypropyl β cyclodextrin, by Merck Group (1 mentions)
Celltracker green 5 chloromethylfluorescein diacetate, by Thermo Fisher Scientific (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
4 protocols using sapogenin
1
Parasite Isolation and Lysis Protocol
2
Cultivation and Bioconversion of B. megaterium
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Either LB- (25 g/L, Becton–Dickinson) or TB- (24 g/L yeast extract, 12 g/L tryptone, 0.4% glycerol, 100 mM potassium phosphate buffer, pH 7.4) media were used for the cultivation of B. megaterium. For the preculture, 50 mL of medium either containing 10 µg/mL tetracycline (cells harboring a variant of pSMF2.1) or 10 µg/mL chloramphenicol (cells harboring a variant of pSMF3) were inoculated with cells from an agar plate or glycerol stock. For the main culture, 50 mL of medium containing 10 µg/mL of the required antibiotic were inoculated with 500 µL of the preculture. The main culture was grown until an optical density of ~0.4 was reached, protein expression was induced by addition of 0.25 g xylose dissolved in 1 mL distilled water. For whole-cell conversion experiments, substrates were dissolved in a 45% 2-hydroxypropyl-β-cyclodextrin/4% Quillaja saponin-solution. 2.5 mL of the solution were added to the culture directly after protein induction. A final substrate concentration of 300 µM was used for all conversion experiments. Cholesterol from sheep wool (purity ≥99%), 7-dehydrocholesterol (≥98%), β-sitosterol from plant extracts (purity ≥70%, containing residual campesterol), 2-hydroxypropyl-β-cyclodextrin (average molecular weight ~1,460) and Quillaja saponin (Sapogenin content ≥10%) were purchased from Sigma-Aldrich.
3
Antibody-based Oxidative Stress Assessment
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Anti-human 4-hydroxynonenal (HNE)-Michael adduct antibody and anti-human dinitrophenyl (DNP) antibody were obtained from Calbiochem (La Jolla, CA, USA). DCF-DA (2,7-dichlorodihydrofluorescein diacetate) and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). CellTracker Green 5-chloromethylfluorescein diacetate (CMFDA) was purchased from Molecular Probes (Life Technologies, Carlsbad, CA, USA). Saponin containing 8–25 wt % of sapogenin was purchased from Sigma–Aldrich Co. (St. Louis, MO, USA). Vitamin A (VA; minimum 1,000,000 IU vitamin A/g, containing 55.5% of vitamin A palmitate, 43.5% of peanut oil, and 1% of dl -α-tocopherol) and vitamin E-acetate (VE; 1100 IU vitamin E/g, containing 100% of dl -α-tocopherol) were purchased from DSM (Heerlen, The Netherlands). Double-distilled water was used for all solutions and emulsions.
4
Quantifying Total Saponins Content
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The total saponins content (TSC) was determined using a vanillin-glacial acetic acid colorimetric method as described in previous studies with some modifications (Chen et al., 2007 (link); Yan et al., 2011 (link)). The plant extracts (625 μg) were transferred to test tubes and mixed with 0.2 mL freshly prepared 5% (w/v) vanillin-acetic acid solution. Perchloric acid (1.2 mL) was added and the mixture was incubated at 65°C for 15 min. After the mixture was cooled on ice, acetic acid was added to a final volume of 5 mL, then mixed and centrifuged. The absorbance of the supernatant was scanned at 557 nm by UV-VIS spectrophotometer. A saponin standard (containing 20–35% sapogenin, Sigma-Aldrich), in the range of 0.25–2 mg, was used as the standard equivalent, and TSC of the samples was calculated from the calibration curve and expressed as percentage saponins equivalents per gram of dried weight (% w/w).
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