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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-SS-biotin (Succinimidyl-2-(biotinamido)-ethyl-1,3-dithiopropionate) is a long-chain cleavable amine-reactive biotinylation reagent that has a spacer arm of 24.3 A in length due to the existence of a cross-bridge within its chemical structure. The long spacer arm creates an increased length between a covalently modified molecule and the bicyclic biotin rings that greatly enhances the ability of the molecule to bind to avidin or streptavidin probes. The NHS ester group of NHS-SS-biotin reacts with the amine groups of proteins and other molecule forming an amide bond and releasing of sulfo-NHS. NHS-SS-Biotin is a water-insoluble molecule that requires the dissolution of organic solvents prior to be added to aqueous reactions.
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 • Cleavable—disulfide bond in spacer arm allows the biotin label to be removed using reducing agents such as DTT • Solubility—water insoluble, must be dissolved in DMSO or DMF before further dilution in aqueous buffers • Medium length—spacer arm is 24.3 angstroms; it consists of the native biotin valeric acid group extended by a 7-atom chain
Sample
hippocampal neurons
Preparation method
Soluble in DMSO or DMF.
Reaction Conditions
1.5 mg/ml, 4 ℃ for 1 h
Applications
Neurons were washed with the artificial cerebrospinal fluid（ACSF） at 37 ℃, and incubated with 1.5 ml of 1.5 mg/ml NHS-SS-biotin with gentle shaking at 4 ℃ for 1 h. After washing, neurons were switched to the neuronal culture medium and incubated at 37 ℃. At indicated times of incubation, neurons were cooled to 4 ℃ and un-endocytosed surface biotin was cleaved by incubating in the glutathione cleavage buffer (50 mM glutathione, 75 mM NaCl, 10 mM EDTA, 1% BSA, and 0.075 N NaOH). Neurons were lysed in the modified RIPA buffer (50 mM Tris–HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxylate, 1 mM EDTA, and protease inhibitors). Lysates were cleared by centrifugation at 10,000g for 10 min at 4 ℃ and incubated at 4 ℃ over-night with 70 μl of 50% streptavidin beads. Endocytosis of ErbB proteins was assayed using cleavable biotin. Bead-associated proteins were subjected to Western blot analysis.
References:
[1]. Yu Liu, Yan-Mei Tao, Ran-Sook Woo, Wen-Cheng Xiong, Lin Mei. Stimulated ErbB4 internalization is necessary for neuregulin signaling in neurons. Biochemical and Biophysical Research Communications 354 (2007) 505–510.
