The cell-free supernatants were dialyzed against water (cutoff 3,500 Da) and lyophilized, and subsequently hydrolyzed with 5% aqueous CH3COOH (100°C, 3 h). The resulting suspensions were centrifuged (7,000 rpm, 4°C, 30 min), and the supernatant portions were then fractionated on a Sephacryl S-400 HR column (Sigma, 1.5 × 95 cm, flow 16.8 ml/h, fraction volume 2.5 ml), eluted with 0.05 M ammonium hydrogen carbonate. The obtained polysaccharides were further analyzed by chemical analyses.
Sephacryl s 400 hr column
Manufactured by Merck Group
Sourced in United States
Sephacryl S-400 HR is a size-exclusion chromatography column used for the separation and purification of macromolecules, such as proteins, nucleic acids, and protein complexes, based on their molecular size and shape. The column is made of a cross-linked allyl dextran matrix, which provides a high degree of chemical and physical stability.
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Lab products found in correlation
3 protocols using sephacryl s 400 hr column
1
Polysaccharide Fractionation and Analysis
2
Extraction and Characterization of β-Glucan from P. baumii
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The β-glucan fraction (BGF) was obtained from the carpophores of P. baumii as described previously [16 (link)]. Briefly, dried and powdered carpophores were extracted with distilled water at 100 °C for 10 h. The filtered extract was concentrated in vacuo, and three volumes of ethanol were added at 4 °C for 4 h to precipitate the crude BGF. The crude BGF was purified by passing through a Sephacryl S-400 HR column (Sigma-Aldrich, St. Louis, MO, USA) and BGF was obtained as a strong single band. The β-glucan content in the BGF was 84% by analysis with a mushroom and yeast assay kit (Megazyme, Bray, Ireland). β-Glucans of yeast zymosan A from Saccharomyces cerevisiae and barley were obtained from Sigma-Aldrich. Mouse diets containing 1% normal and 10% high-salt were obtained from Samtako (Osan, Korea).
The polymerase chain reaction (PCR) primers for corin and GAPDH were synthesized by Cosmogenetech (Seoul, Korea). The forward and reverse primer sequences (5′→3′) of corin were 5′GTC CGC ATT ATT CCT CTG GA3′ and 5′ CAA ACC AGA GGA CCA CCA CT3′, respectively. Those of GAPDH were 5′ ACC GCA GCT AGG AAT AAT GGA ATA 3′ and 5′ CTT TCG CTC TGG TCC GTC TT 3′, respectively. Oligo deoxythymine, 5X reaction buffer, Moloney murine leukemia virus (M-MLV) reverse transcriptase, and SYBR green qPCR PreMIX were obtained from Enzynomics (Daejeon, Korea).
The polymerase chain reaction (PCR) primers for corin and GAPDH were synthesized by Cosmogenetech (Seoul, Korea). The forward and reverse primer sequences (5′→3′) of corin were 5′GTC CGC ATT ATT CCT CTG GA3′ and 5′ CAA ACC AGA GGA CCA CCA CT3′, respectively. Those of GAPDH were 5′ ACC GCA GCT AGG AAT AAT GGA ATA 3′ and 5′ CTT TCG CTC TGG TCC GTC TT 3′, respectively. Oligo deoxythymine, 5X reaction buffer, Moloney murine leukemia virus (M-MLV) reverse transcriptase, and SYBR green qPCR PreMIX were obtained from Enzynomics (Daejeon, Korea).
3
Jackfruit Polysaccharide Extraction and Purification
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Jackfruits were obtained from the Xinglong Tropical Botanical Garden, Wanning, Hainan, China. JFP-Ps were extracted according to our previously reported method [15 (link)]. Jackfruits were collected at full maturity (14–16 weeks after flowering) and processed by dicing their flesh, followed by homogenization in a grinder. Subsequently, the homogenate was treated with 80% ethanol for a 24-hour period to remove non-target components. The dried material was dissolved in ultrapure water (material to liquid ratio, 1:30 mL/g) and subjected to extraction at 90 °C for 2.5 h in a water bath. The resultant aqueous extract was concentrated using a rotary evaporator at 55 °C under reduced pressure and subsequently filtered. Ethanol was added to precipitate the mixture at 4 °C overnight. The precipitate was redissolved in ultrapure water, and proteins were removed employing the Sevag method. Following a 72-hour dialysis, the solution underwent chromatographic separation and purification using a Sephacryl™ S-400 HR column (Sigma Chemical Co., St. Louis, MO, USA).
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