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NHS-LC-Biotin Sale

(Synonyms: 生物素化-epsilon-氨基己酸-N-羟基丁二酰亚胺活化酯,Succinimidyl 6-(biotinamido)hexanoate) 目录号 : GC12583

Biotinylation reagent

NHS-LC-Biotin Chemical Structure

Cas No.:72040-63-2

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Sample solution is provided at 25 µL, 10mM.

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实验参考方法

Biotinylation method [1]:

Sample

S. aureus

Preparation method

Soluble in DMSO or DMF.

Reaction Conditions

60 mg/ml, room temperature for 1 h.

Applications

Fixed cell suspensions of S. aureus were diluted to a 2% cell suspension in 0.2 M NaHCO3, pH 8.3. NHS-LC-biotin freshly dissolved in DMSO at a concentration of 60 mg/ml, was added to the cell suspension subsequently. After gentle tumble mixing for 60min at room temperature, the reaction was stopped by removal of excess biotin via gel filtration over a PD-10 column and washed extensively in 0.01 mM Tris-buffered 0.15 M NaCl (TBS). Successful protein biotinylation was examining their reactivity with alkaline phosphatase-conjugated streptavidin(AP-STRAV). AP-STRAV binding was detected as a function of pNPP hydrolysis.

References:

[1]. TRUC NGUYEN, BERHANE GHEBREHIWET, AND ELLINOR I. B. PEERSCHKE. Staphylococcus aureus Protein A Recognizes Platelet gC1qR/p33: a Novel Mechanism for Staphylococcal Interactions with Platelets. INFECTION AND IMMUNITY 2000, p. 2061–2068.

产品描述

NHS-LC-biotin (succinimidyl-6-(biotinamindo)hexanoate), also known as NHS-X-biotin is a derivative of D-biotin, a amine-reactive biotinylation agent, that contains a spacer arm off the valeric acid side chain of D-biotin with an NHS ester group at its end. The NHS ester group at the end of NHS-LC-biotin covalently binds to amine groups in proteins and other molecules forming a stable amide linkage and releasing the NHS group. The 6-aminocaproic acid spacer of NHS-LC-biotin greatly increases the length between a covalently modified molecule and the bicyclic biotin rings leading to a better binding potential for avidin or streptavidin probes. NHS-LC-biotin is insoluble in aqueous environments requiring the dissolution of organic solvents prior to the addition to a buffered reaction.

Reference

[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.

Chemical Properties

Cas No. 72040-63-2 SDF
别名 生物素化-epsilon-氨基己酸-N-羟基丁二酰亚胺活化酯,Succinimidyl 6-(biotinamido)hexanoate
化学名 (2,5-dioxopyrrolidin-1-yl) 6-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]hexanoate
Canonical SMILES C1CC(=O)N(C1=O)OC(=O)CCCCCNC(=O)CCCCC2C3C(CS2)NC(=O)N3
分子式 C20H30N4O6S 分子量 454.54
溶解度 ≥ 22.727mg/mL in DMSO 储存条件 Store at -20°C, protect from light, unstable in solution, ready to use.
General tips 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液;一旦配成溶液,请分装保存,避免反复冻融造成的产品失效。
储备液的保存方式和期限:-80°C 储存时,请在 6 个月内使用,-20°C 储存时,请在 1 个月内使用。
为了提高溶解度,请将管子加热至37℃,然后在超声波浴中震荡一段时间。
Shipping Condition 评估样品解决方案:配备蓝冰进行发货。所有其他可用尺寸:配备RT,或根据请求配备蓝冰。

溶解性数据

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1 mg 5 mg 10 mg
1 mM 2.2 mL 11.0001 mL 22.0003 mL
5 mM 0.44 mL 2.2 mL 4.4001 mL
10 mM 0.22 mL 1.1 mL 2.2 mL
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动物体内配方计算器 (澄清溶液)

第一步:请输入基本实验信息(考虑到实验过程中的损耗,建议多配一只动物的药量)
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Research Update

Improvement of an EIA system for basic fibroblast growth factor by use of biotinylated antibody prepared with NHS-LC-Biotin

J Clin Lab Anal 1995;9(2):96-100.7714669 10.1002/jcla.1860090204

We improved our previously devised enzyme immunoassay (FIA) system for basic fibroblast growth factor (bFGF) using biotinylated antibody prepared with sulfosuccinimidyl-6-(biotinamido)hexanoate (NHS-LC-Biotin, Pierce), a water-soluble biotin analogue, with an extended spacer arm. The discriminatory detection limit of the improved EIA was found to be 5 pg/ml (0.5 pg/assay tube), sixfold more sensitive than that of the previous system. The reproducibility of within- and between-assay series was 5.10-8.61% and 5.99-8.69%, respectively; and recovery of exogenous bFGF from serum was approximately 102%. Employing the improved EIA system, we investigated by chromatofocusing chromatography the isoelectric points of two immunoreactive bFGFs (high-molecular-weight bFGF-like immunoreactive substance, designated as HMW-bFGF-LI and 16-kd bFGF-LI, having the same molecular weight as recombinant bFGF) detected in serum from a breast cancer patient. As a result, the pI value of HMW-bFGF-LI was estimated to be 7.13, and that of 16 kd bFGF-LI, 9.58.

In vivo labeling of Escherichia coli cell envelope proteins with N-hydroxysuccinimide esters of biotin

Appl Environ Microbiol 1993 Mar;59(3):663-8.8480997 PMC202171

The primary amine coupling reagents succinimidyl-6-biotinamido-hexanoate (NHS-A-biotin) and sulfosuccinimidyl-6-biotinamido-hexanoate (NHS-LC-Biotin) were tested for their ability to selectively label Escherichia coli cell envelope proteins in vivo. Probe localization was determined by examining membrane, periplasmic, and cytosolic protein fractions. Both hydrophobic NHS-A-biotin and hydrophilic NHS-LC-Biotin were shown to preferentially label outer membrane, periplasmic, and inner membrane proteins. NHS-A- and NHS-LC-Biotin were also shown to label a specific inner membrane marker protein (Tet-LacZ). Both probes, however, failed to label a cytosolic marker (the omega fragment of beta-galactosidase). The labeling procedure was also used to label E. coli cells grown in low-salt Luria broth medium supplemented with 0, 10, and 20% sucrose. Outer membrane protein A (OmpA) and OmpC were labeled by both NHS-A- and NHS-LC-Biotin at all three sucrose concentrations. In contrast, OmpF was labeled by NHS-A-biotin but not by NHS-LC-Biotin in media containing 0 and 10% sucrose. OmpF was not labeled by either NHS-A- or NHS-LC-Biotin in E. coli cells grown in medium containing 20% sucrose. Coomassie-stained gels, however, revealed similar quantities of OmpF in E. coli cells grown at all three sucrose concentrations. These data indicate that there was a change in outer membrane structure due to increased osmolarity, which limits accessibility of NHS-A-biotin to OmpF.

A novel intravascular drug delivery method using endothelial biotinylation and avidin-biotin binding

Drug Deliv 2001 Oct-Dec;8(4):215-22.11757779 10.1080/107175401317245895

In this study, a novel intravascular drug delivery system was developed in which a drug injected from a catheter was fixed to the vasculature of the targeted tissue. Cellular proteins of viable endothelial cells were first biotinylated directly by biotinylation reagents, and then bound by an avidinated drug or, using avidin as a linker, a biotinylated drug. In the initial experiments, we studied in vitro the biotinylation of cultured bovine aortic endothelial cells (BAECs) by applying biotinylation reagents (NHS-LC-Biotin or sulfo-NHS-LC-biotin) onto the washed intact BAEC monolayers and showed that the amount of biotin bound to the cells depended on the concentration of the biotinylation reagents applied. The cell-bound biotin decreased with time after the biotinylation. When fluorescein-labeled avidin (FITC-avidin) was applied to the biotinylated BAEC monolayers, the FITC-avidin readily bound to the cells. An LDH-release assay showed that sulfo-NHS-LC-biotin was only slightly cytotoxic to the BAECs and a colony formation assay showed only slight adverse effects of the reagent. In vivo studies were carried out on the renal arteries of normal rabbits. A solution of NHS-LC-Biotin was injected through a catheter to one kidney to biotinylate its vasculature and the vehicle to the other as control, followed by a perfusion with saline. Finally, a solution of FITC-avidin was injected to both kidneys that were then reperfused with the blood flow following the withdrawal of the catheters. In the histological sections, more than 85% of glomeruli was stained with fluorescein in the biotinylated kidney, whereas no glomeruli were stained in the control. In the kidneys harvested 2 days after the same procedure, most glomeruli were still brightly stained. In the final experiment, biotinylated kidneys were injected with a solution of avidin, followed by a solution of fluorescein-biotin. Control kidneys had no prior biotinylation but received the same injections of avidin and fluorescein-biotin as above. More than 80% of glomeruli were stained in the biotinylated kidneys but none in the controls. This indicated that biotinylated drugs can be anchored to the biotinylated vasculature through avidin without being flushed away by blood flows. No apparent adverse effect was found in the functions of biotinylated kidneys. We propose that this drug delivery system is feasible for the treatment of some pathological conditions of blood vessels such as microvascular proliferation in malignant tumors and for continuous drug delivery in certain target organs.

Identification of surface components of mammalian respiratory tract cilia

Cell Motil Cytoskeleton 1990;17(4):317-28.2076548 10.1002/cm.970170407

Cilia isolation methods were modified to retain respiratory tract ciliary membranes and to identify accessible surface components. Prior to isolation of cilia, halves of cow tracheae were treated with the extended spacer arm analog of N-hydroxysuccinimido-biotin (NHS-LC-Biotin) to label accessible membrane constituents. Mechanical disruption of the epithelium and substitution of CHAPS for Triton X-100 provided a good yield of cilia with membranes and with minimal contamination. Subsequent extraction of these cilia with Triton X-100 solubilized the membranes and released soluble matrix proteins. Proteins of membrane + matrix and axoneme fractions were analyzed after electrophoresis in sodium dodecyl sulfate polyacrylamide gels. The major biotin-labeled components in the membrane + matrix fraction were 105, 98, and 92 kd, were glycosylated, and remained with reconstituted, pelleted membrane vesicles along with the major non-biotinylated protein at 51 kd. Other membrane + matrix proteins at 126 and 76 kd bound streptavidin even from nonlabeled trachea, but remained soluble. Several biotin-labeled proteins distinct from those in the membrane fraction remained with Triton X-100-extracted axonemes. Streptavidin-colloidal-gold (SAG) particles appeared to bind randomly along the length of cilia. The peripheral join between A and B microtubules was a predominant nonspecific location of SAG on axonemes. Axonemes with biotin label also bound significant numbers of SAG to outer dynein arms, confirming the streptavidin reaction with separated proteins on transfers. These results suggest close association of the membrane with the axoneme in respiratory tract cilia and a membrane composition somewhat different from protozoan cilia.

Biotinylation of membrane proteins accessible via the pulmonary circulation in normal and hyperoxic rats

Am J Physiol 1997 Mar;272(3 Pt 1):L461-70.9124603 10.1152/ajplung.1997.272.3.L461

It is well established that the phenotype of the pulmonary vascular surface can be affected by injurious stimuli, but the few proteins for which the expression and/or activity have been studied make up only a small fraction of the entire spectrum of luminal cell membrane proteins. To expand the capability for studying such proteins, we developed a method for biotinylating cell membrane proteins accessible via the vascular lumen in the isolated-perfused rat lung and examined the impact of hyperoxia on the spectrum of the biotinylated proteins. Labeling was carried out either by single-pass bolus injection of the cell impermeant biotinylation reagent sulfosuccinimidyl 6-biotin-amido hexanoate (NHS-LC-Biotin) into the pulmonary artery cannula or by the addition of NHS-LC-Biotin to a lung homogenate. Lung membrane fractions were prepared, and the proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose by electroblotting. The biotinylated proteins were visualized using a chemiluminescent substrate for streptavidin-linked horseradish peroxidase. The spectrum of proteins biotinylated via the vasculature was distinct from that of the biotinylated lung homogenate. Lectin affinity purification of biotinylated proteins from the lung membrane fractions of normal lungs biotinylated via the vasculature revealed characteristic spectra that were reproducibly different from those from rats exposed to hyperoxia for 48-60 h. These results demonstrate that biotinylation of membrane proteins accessible to an extracellular reagent during a single transit through the pulmonary vascular bed is feasible and that the spectrum of these labeled proteins reveals the effects of hyperoxic lung injury. The affinity of biotin for streptavidin makes this procedure potentially useful as a means of separating the labeled membrane proteins from the much larger population of membrane proteins that are not accessible via the vasculature, e.g., intracellular membrane proteins and plasma membrane proteins of cell types in luminally inaccessible regions of the intact lung. The consistent changes in the spectrum of labeled proteins seen with hyperoxia suggest that in itself the spectrum may be a useful encryption of certain aspects of vascular pathophysiology.