Amino-dextran was synthesized as previously reported (Nakamura et al., 2010 (link)). The synthesis and chemical characterization of dextran conjugates were described in details in our previous paper (Morimoto et. al., Bioconjugate Chem. 2014). In short, dextran (500 mg, average molecular weight 35,000–45,000, from Leuconostoc mesenteroids, Sigma) was dissolved in 50 mL of anhydrous DMF and treated with 126 mg of N,N′-carbonyldiimidazole and 250 µL of ethylenediamine. Aminodextran was treated with biotin-NHS ester in DMSO at 4 °C for overnight to conjugate biotin. COPA or peptide ligands were conjugated to the biotinylated-dextran polymer through N-(α-Maleimidoacetoxy) succinimide ester (AMAS, from Thermo scientific) in DMSO for two hours in the dark. The unreacted reactants were removed by filtering through Amicon Ultra 4 mL centrifugal filter unit (10,000 NMWL, from Millipore). The ligand-dextran conjugate in the filter unit was recovered in 1×PBS according to the manufacturer's protocol. The concentration of biotin in the solution was quantitatively determined using the Fluorescence Biotin Quantitation Kit (Thermo Scientific). The procedure to estimate the number of ligands conjugated to dextran polymer is described in details in Supplemental experimental procedures .
Leuconostoc mesenteroids
Manufactured by Merck Group
Leuconostoc mesenteroids is a gram-positive, catalase-negative, heterofermentative bacterium. It is commonly used in the production of various fermented foods and beverages. This organism plays a role in the conversion of sugars, producing lactic acid and carbon dioxide as byproducts.
Automatically generated - may contain errors
Lab products found in correlation
N α maleimidoacetoxy succinimide ester amas, by Thermo Fisher Scientific (1 mentions)
Amicon ultra 4 ml centrifugal filter unit, by Merck Group (1 mentions)
Fluorescence biotin quantitation kit, by Thermo Fisher Scientific (1 mentions)
Snakeskin dialysis tubing, by Thermo Fisher Scientific (1 mentions)
2 protocols using leuconostoc mesenteroids
1
Synthesis and Characterization of Biotin-Dextran Conjugates
2
Synthesis of Amino-Dextran Conjugates
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Amino-dextran was prepared
as previously reported.35 (link) 500 mg of dextran
(average molecular weight 35 000–45 000, from Leuconostoc mesenteroids, Sigma-Aldrich) was dissolved in
anhydrous DMSO in 50 mL tube. The solution was warmed to 50 °C
to completely dissolve the dextran. 126 mg of N,N′-carbonyldiimidazole in 500 μL of anhydrous
DMSO was added and the solution was incubated with gentle shaking
at 50 °C for 20 min. After the incubation, 250 μL of ethylenediamine
was added and the solution was incubated with gentle shaking at 50
°C for 22 h. 25 mL of acetone was added and the solution was
cooled on ice for 15 min. The tube was centrifuged at 700g for 10 min and the supernatant was removed. The pellet was resuspended
in 20 mL of acetone and the tube was centrifuged at 700g for 10 min. The supernatant was removed and the pellet was air-dried.
The dried pellet was dissolved in 10 mL of ultrapure water and the
solution was dialyzed with Snakeskin dialysis tubing (7 MWCO, Thermo
Scientific) for 2 days. The dialyzed dextran was lyophilized and used
for peptoid/peptide conjugation. The extent of amine derivatization
was determined by a colorimetric test using 2,4,6-trinitrobenzenesulfonic
acid (TNBS).43 (link)
as previously reported.35 (link) 500 mg of dextran
(average molecular weight 35 000–45 000, from Leuconostoc mesenteroids, Sigma-Aldrich) was dissolved in
anhydrous DMSO in 50 mL tube. The solution was warmed to 50 °C
to completely dissolve the dextran. 126 mg of N,N′-carbonyldiimidazole in 500 μL of anhydrous
DMSO was added and the solution was incubated with gentle shaking
at 50 °C for 20 min. After the incubation, 250 μL of ethylenediamine
was added and the solution was incubated with gentle shaking at 50
°C for 22 h. 25 mL of acetone was added and the solution was
cooled on ice for 15 min. The tube was centrifuged at 700g for 10 min and the supernatant was removed. The pellet was resuspended
in 20 mL of acetone and the tube was centrifuged at 700g for 10 min. The supernatant was removed and the pellet was air-dried.
The dried pellet was dissolved in 10 mL of ultrapure water and the
solution was dialyzed with Snakeskin dialysis tubing (7 MWCO, Thermo
Scientific) for 2 days. The dialyzed dextran was lyophilized and used
for peptoid/peptide conjugation. The extent of amine derivatization
was determined by a colorimetric test using 2,4,6-trinitrobenzenesulfonic
acid (TNBS).43 (link)
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