Description:
DBCO (Dibenzocyclooctyne) is a cycloalkyne that can reacts with azides via strain-promoted 1,3-dipolar cycloaddition in aqueous solution, a bioorthogonal reaction also termed Cu-free click reaction. This reaction has excellent selectivity and biocompatibility, such that the complimentary reagents can form covalent bonds with richly functionalized biological systems. The Cu-free click reaction has been a powerful tool in catalyst-free bioconjugation. DBCO reagents posses fast kinetcs and stability in aqueous buffer and they can be used to label azide-modified biomolecules with high specificity and reactivity.
Physical Properties:
- Yellow/pale-yellow solid or viscous liquid;
- Soluble in DMSO or DMF;
Storage Conditions:
- Store at -20 0C, desiccate. NHS tends to hydrolyze from moisture. Avoid frequent thaw and frozen.
Reaction Procedures:
NHS esters are moisture senstivie. To avoid moisture condensation onto the product always let vial come to room temperature before opening; be exposure to limit exposure to moisture and restore under atmosphere. The NHS ester moiety readily hydrolyzes and becomes non-reactive; therefore, prepare stock solutions immediately before use. Stock solutions in anhydrous solvents can be kept for several days (freeze when not in use). Hydrolysis of the NHS ester is a competing reaction. Conjugation with primary amines of proteins/peptides (i.e., acylation) is favored at near neutral pH (6-9) and with concentrated protein solutions. For conjugation, use non-amine containing buffers at pH 7-9 such as PBS (20 mM sodium phosphate, 150 mM sodium chloride, pH 7.4); 20 mM HEPES; 100 mM carbonate/biocarbonate; or 50 mM borate buffer. Do not use buffers that contain primary amines, (e.g., Tris, glycine). Avoid buffers that contain azides, which can react with DBCO. Dissolve DBCO water miscible organic solvent such as DMSO or DMF before diluting in final reaction buffer. DBCO-NHS ester is not soluble in aqueous buffers.
Materials Required:
- Conjugation buffer: Sodium bicarbonate 100 mM buffer, pH 8.5 or other amine-free buffer at pH 7-8.5.
- Water miscible solvents: DMSO or DMF.
- Quenching buffer: 1 M Tris.HCl, pH 8.0.
Reaction Steps:
Prepare proteins in PBS. Immediately before use, prepare 10 mM of the DBCO-NHS reagent in DMSO or DMF. Add the NHS reagent to the protein sample at a final: mM. If For samples <5 mg/ml, use a 20 to 50 fold molar excess. Incubate the reaction at room temperature for 30 minutes or on ice for 2 hours. Stop the reaction by adding Quenching Buffer 100 mM Tris.or on ice for 15 minutes. Remove non reagentby dialysis or desalting.
Copper-free Click Reaction:
Prepare the azide-containing sample in reaction buffer. Add DBCO conjugate to azide containing sample. Recommendation: Add 1 mole equivalent of limiting reagent to 1.5-3.0 mole equivalents of highest abundance reagent. Incubate the reaction at room temperature for 2-4 hours or 2-12 hours at 4 degree celcius. The reaction is now ready for purification.
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修饰性PEG的用途
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bhclna2so4nano3na2co3
chclnaohna2co3nacl
dba(oh)2nahco3alcl3nahso4
通过解答,教会我,谢谢
(1)优级纯试剂 亦称保证试剂,为一级品,纯度高,杂质极少,主要用于精密分析和科学研究,常以GR表示。
(2)分析纯试剂 亦称分析试剂,为二级品,纯度略低于优级纯,杂质含量略高于优级纯,适用于重要分析和一般性研究工作,常以AR表示。
(3)化学纯试剂 为三级品,纯度较分析纯差,但高于实验试剂,适用于工厂、学校一般性的分析工作,常以CP表示。
(4)实验试剂 为四级品,纯度比化学纯差,但比工业品纯度高,主要用于一般化学实验,不能用于分析工作,常以 LR表示。
以上按试剂纯度的分类法已在我国通用。根据化学工业部颁布的“化学试剂包装及标志”的规定,化学试剂的不同等级分别用各种不同的颜色来标志,见表1。
表1 我国化学试剂的等级及标志
A.H2SO4Na2SO4AgNO3BaCl2.
B.NaOHNa2CO3NaHSO4MgCl2
C.CaCl2NaNO3MgSO4BaCl2
D.HNO3KOHKClK2CO3
A、Na2CO3HClH2SO4NaNO3
B、K2CO3H2SO4HNO3BaCl2
C、HClHNO3AgNO3NaCl
D、NaOHFeCl3MgCl2BaCl2