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 .
Amicon ultra 4 ml centrifugal filter unit
Manufactured by Merck Group
Sourced in United States
The Amicon Ultra 4 mL centrifugal filter unit is a laboratory instrument designed for sample concentration and buffer exchange. It features a regenerated cellulose membrane with a specific molecular weight cutoff to selectively retain molecules of interest while allowing smaller molecules to pass through during the centrifugation process.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescence biotin quantitation kit, by Thermo Fisher Scientific (1 mentions)
Leuconostoc mesenteroids, by Merck Group (1 mentions)
N α maleimidoacetoxy succinimide ester amas, by Thermo Fisher Scientific (1 mentions)
Luminescent image analyser las 4000, by Fujifilm (1 mentions)
Amicon ultra 0.5 ml, by Merck Group (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Low range ssrna ladder, by New England Biolabs (1 mentions)
Sybr green 2, by Lonza (1 mentions)
2 protocols using amicon ultra 4 ml centrifugal filter unit
1
Synthesis and Characterization of Biotin-Dextran Conjugates
2
Denaturing PAGE for RNA Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Enzymatic reactions were analysed by denaturing PAGE [4–8% polyacrylamide (acrylamide/N, N′-methylenebisacrylamide, 19:1), 7.5 M urea, 25% (v/v) formamide, 89 mM Tris, 89 mM boric acid, 2 mM EDTA]. The RNA samples were mixed with equal amount of 2× formamide loading solution [80% (v/v) formamide, 10 mM EDTA pH 8.0, 0.1 mg/mL xylene cyanol FF, 0.1 mg/mL bromophenol blue], and heated at 90 °C for 3 min before being loaded on the gel. The gels were electrophoresed and stained with SYBR Green II (Lonza) and visualised on a BioRad ChemiDoc XRS+ System (BioRad, Hercules, CA, USA), Luminescent Image Analyser LAS 4000 (Fujifilm, Tokyo, Japan) or FAS-IV Imaging System (Nippon Genetics, Tokyo, Japan). Low Range ssRNA Ladder (New England Biolabs, Ipswich, MA, USA) was used as the size marker. RNAs were also purified by preparative denaturing PAGE. The RNA bands were visualised by UV shadowing, and crushed and extracted with water. The extracts were desalted by centrifugation with Amicon Ultra-0.5 mL or Amicon Ultra-4 mL centrifugal filter unit (Millipore, Billerica, MA, USA), according to the manufacturer’s instructions. The RNAs were then precipitated with sodium acetate (pH 5.2) and 2-propanol.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!