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In vitro transcription of capped mRNA with modified nucleotides and Poly(A) tail
TSA (Tyramide Signal Amplification), used for signal amplification of ISH, IHC and IC etc.
Separation of phosphorylated and non-phosphorylated proteins without phospho-specific antibody
A convenient and sensitive way for cell proliferation assay and cytotoxicity assay
Protect the integrity of proteins from multiple proteases and phosphatases for different applications.
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
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.
• Protein labeling—biotinylate antibodies or other proteins for detection or purification using streptavidin probes or resins • Intracellular labeling—membrane-permeable, can be used to label inside cells • Amine-reactive—reacts with primary amines (-NH2), such as the side-chain of lysines (K) or the N-terminal-amine • Irreversible—forms permanent amide bonds; spacer arm cannot be cleaved • Solubility—water insoluble, must be dissolved in DMSO or DMF before further dilution in aqueous buffers • Medium length—spacer arm is 22.4 angstroms; it consists of the biotin valeric acid group extended by a 6-atom chain
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.