Biotin-HPDP

A sulfhydryl-reactive biotinylation reagent

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货号: ajci6316
CAS: 129179-83-5
别名: N-(6-[生物素胺]己基)-3-(2-吡啶二硫)丙酰胺,BiotinHPDP
分子式: C124H37N5O3S3
分子量: 539.78
纯度: >98%
溶解度: ≥ 101.4mg/mL in DMSO, ≥ 8.29 mg/mL in EtOH with ultrasonic
储存: Store at -20°C
库存: 现货

Background

Biotin-HPDP is a sulfhydryl-reactive biotinylation reagent that forms a reversible disulfide linkage.Biotin-HPDP consists of a bicyclic Biotin ring structure, a 1, 6-diaminohexane attached to the side chain of biotin valonate, and a sulfhydryl reactive group at the end of the side chain^[1]. It is used to label protein cysteines and other substrates that contain sulfhydryl groups.

After NEM alkylation, certain (but not all) residual free cysteine residues may react with biotin-HPDP, resulting in the coisolation of proteins containing such cysteine residues^[2].

A kinetic study of the modification reactions that generate monothiophosphate disulfide linkages with either 5'-GMPS alone or 5'-GMPS-primed RNA as the substrate revealed that the second-order rate constants increased as the pH was decreased. For example, when the reaction pH was lowered from 8 to 4, the k2 value for the coupling reaction between N-(6-[biotinamido]hexyl)-3'-(2'-pyridyldithio)propionamide (biotin-HPDP) and GMPS increased 67-fold from 1.84 to 123 M(-1) x s(-1) ^[3].

参考文献:
[1]: Bioconjugate Techniques , 2nd ed. By Greg T.Hermanson ?(Pierce Biotechnology, Thermo Fisher Scientific, Rockford, IL). ?Academic Press ?(an imprint of Elsevier): ?London, Amsterdam, Burlington, San Diego . 2008. ISBN 978-0-12-370501-3.
[2]: Zhou B, Wang Y, Yan Y, Mariscal J, Di Vizio D, Freeman MR, Yang W. Low-Background Acyl-Biotinyl Exchange Largely Eliminates the Coisolation of Non-S-Acylated Proteins and Enables Deep S-Acylproteomic Analysis. Anal Chem. 2019 Aug 6;91(15):9858-9866. doi: 10.1021/acs.analchem.9b01520. Epub 2019 Jul 11. PMID: 31251020; PMCID: PMC7451198.
[3]: Wu CW, Eder PS, Gopalan V, Behrman EJ. Kinetics of coupling reactions that generate monothiophosphate disulfides: implications for modification of RNAs. Bioconjug Chem. 2001 Nov-Dec;12(6):842-4. doi: 10.1021/bc0100612. PMID: 11716671.

Biotin-HPDP 是一种巯基反应性生物素化试剂，可形成可逆的二硫键。Biotin-HPDP 由双环生物素环结构、连接到生物素戊酸酯侧链的 1, 6-二氨基己烷和一个巯基反应性物质组成组在侧链的末端^[1]。用于标记蛋白质半胱氨酸和其他含有巯基的底物。

NEM 烷基化后，某些（但不是全部）残留的游离半胱氨酸残基可能与生物素-HPDP 反应，导致含有此类半胱氨酸残基的蛋白质共分离^[2]。

对以单独的 5'-GMPS 或以 5'-GMPS 引发的 RNA 作为底物产生单硫代磷酸盐二硫键的修饰反应的动力学研究表明，二级速率常数随着 pH 值的降低而增加。例如，当反应 pH 值从 8 降低到 4 时，N-(6-[生物素氨基]己基)-3'-(2'-吡啶二硫代)丙酰胺（生物素-HPDP）与 GMPS 之间的偶联反应的 k2 值从 1.84 增加 67 倍至 123 M(-1) x s(-1) ^[3]。

Protocol

Biotinylation method [1]:
Sample	S-nitrosylated proteins
Preparation method	Soluble in DMSO or DMF.
Reaction Conditions	50mM, 25 ℃ for 1 h
Applications	Prepare biotin-HPDP as a 50mM suspension in DMSO, Dilute with DMF to a final concentration of 4 mM. Add 1:3 volume of Labeling Solution and 1:50 volume of Ascorbate Solution（50mM） to the blocked protein samples, incubate for 1 hour at 25°C. After that, add two volumes of ?20°C acetone and incubate for 20 min at ?20°C to remove the biotin-HPDP. At last, Add 15 μl of packed streptavidin-agarose per mg of protein used in the initial protein sample, to purify biotinylated proteins. Incubate the biotinylated proteins with the resin for 1 hour at room temperature. Wash the beads five times with 10 volumes of Neutralization Buffer + NaCl. Centrifuge at 200g for 5 s at room temperature between each wash. Incubate the beads with Elution Buffer to recover the bound proteins. To test for the protein of interest with specific antibodies in SDS-PAGE.
参考文献: [1].Samie R. Jaffrey and Solomon H. Snyder. The Biotin Switch Method for the Detection of S-Nitrosylated Proteins. Science’s stke.2015.