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In vitro transcription of capped mRNA with modified nucleotides and Poly(A) tail
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Separation of phosphorylated and non-phosphorylated proteins without phospho-specific antibody
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Protect the integrity of proteins from multiple proteases and phosphatases for different applications.
Sulfo-NHS-SS-biotin(sulfosuccinimidyl-20(biotinamido)ethyl-1,3-dithiopropionate) is a long-chain cleavable amine-reactive biotinylation reagent. The presence of the negatively charged sulfonate group in the chemical structure of sulfo-NHS-SS-biotin makes it a water-soluble biotinylation reagent that can be directly added to aqueous reactions without prior dissolution of organic solvents. Although no prior dissolution is required, an aqueous stock solution of sulfo-NHS-SS-biotin must be prepared rapidly and used immediately in case of the occurrence of hydrolysis of the active ester. Sulfo-NHS-SS-biotin has been used to react with amine-containing proteins and other molecules forming a complex which further interacts with avidin or streptavidin probes and to purify targeted molecules using affinity chromatography on a column of immobilized avidin or streptavidin.
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 to facilitate immobilization, purification or detection • Cell surface labeling—do not penetrate the plasma membrane, biotinylates only surface proteins of whole cells • Amine-reactive—reacts with primary amines (-NH2), such as lysine side-chains, or the N-terminal-amine • Cleavable—disulfide bond in spacer arm allows the biotin label to be removed using reducing agents such as DTT • Soluble—charged sulfo-NHS group increases reagent water solubility compared to ordinary NHS-ester compounds • Medium length—spacer arm is 24.3 angstroms; it consists of the native biotin valeric acid group extended by a 7-atom chain
Sample
468/EV cells and 468/uPAR cells.
Preparation method
Soluble in water, DMSO or DMF.
Reaction Conditions
1 mg/ml, 15minutes on ice
Applications
Cells in monolayer culture (1.5 × 106) were washed three times with ice-cold PBS and then treated with sulfo-NHS-SS-biotin (1mg/mL) for 15 minutes on ice. Biotinylation reactions were terminated with 100 mmol/L glycine in PBS. After washing with PBS, cell extracts were prepared in radioimmunoprecipitation assay (RIPA) buffer (20 mmol/L sodium phosphate, 150 mmol/L NaCl (pH 7.4), 1% NP40, 0.1% SDS, and 0.5% deoxycholic acid) with protease inhibitor cocktail. Biotinylated membrane proteins were precipitated with streptavidin-sepharose. Proteins were eluted with SDS sample buffer, resolved by SDS-PAGE, electrotransferred to polyvinylidene difluoride (PVDF) membranes, and probed with primary antibodies.
References:
[1]. Minji Jo, Boryana M. Eastman, Drue L. Webb, et al. Cell Signaling by Urokinase-type Plasminogen Activator Receptor Induces Stem Cell-like Properties in Breast Cancer cells . Cancer Res, 2010;70:8948-8958