A biotinylation reagent
Biotin hydrazide is a biotinyl derivative that can be used as a probe for the determination of protein carbonylation, which is a component of several diseases. Protein carbonylation, an irreversible post translational modification (PTM), is caused by attack of reactive oxygen species (ROS), numerous lipid oxidation products (such as α,β-unsaturated γ-hydroxyalkenals), or nonemzymatic glycation resulting in the loss of protein function. Biotin hydrazide is a preferred carbonyl-reactive probe for its direct reaction and chemistry and no requirement of additional catalysts or reducing agents. Biotin hydrazide exclusively and readily derivatizes carbonyl groups at pH 5.5, which facilitates its use to measure carbonylated proteins in biological samples.
Reference
[1].Kenneth Hensley, Kelly S. Williamson. Protein Carbonyl Determination Using Biotin Hydrazide. Methods in Biological Oxidative Stress. Methods in Pharmacology and Toxicology 2003, pp 195-199
[2].Madian AG, Regnier FE. Proteomic identification of carbonylated proteins and their oxidation sites. J Proteome Res. 2010;9(8):3766-3780.
| Biotinylation method [1]: | |
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Sample |
human umbilical cord HA |
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Preparation method |
Soluble in DMSO. Dissolve glycoprotein at a concentration of 1-5 mg/mL in cold 100mM Sodium Acetate, pH 5.5. |
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Reaction Conditions |
1 mM, overnight at 4℃ |
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Applications |
One hundred milligrams of human umbilical cord HA was dissolved in 0.1 M Mes, pH 5.0, to a ?nal concentration of 1 mg/ml and allowed to dissolve for at least 24 h at 4℃ prior to the coupling of biotin. Biotin hydrazide was dissolved in DMSO as a stock solution of 100 mM and added to the HA solution to a ?nal concentration of 1 mM. A stock solution of 1-ethyl-3-(3-dimethylamin-opropyl) carbidodiimide (EDAC) was prepared as a 100 mM stock solution in dH2O and added to the HA–biotin solution at a ?nal concentration of 30 mM. This solution was left stirred overnight at 4℃. Unlinked biotin and EDAC were removed after the addition of 4M guanidine–HCl. The dialyzed, biotinylated HA (bHA) was aliquoted and stored at 0207C for up to several months. |
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参考文献: [1]. Gregory I. Frost and Robert Stern. A Microtiter-Based Assay for Hyaluronidase Activity Not Requiring Specialized Reagents. ANALYTICAL BIOCHEMISTRY 251, 263–269 (1997). |
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