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EZ-Link Sulfo-NHS-LC-Biotin FAQ


Answers to frequently asked questions (FAQs) about EZ-Link Sulfo-NHS-LC-Biotin


 

What groups does Sulfo-NHS-LC-Biotin react with for biotinylation?
The NHS ester on this biotinylation reagent reacts with primary amines. In proteins and peptides, these occur at the amino terminus and the epsilon amine (side chain) of lysine residues.

What molar excess of Sulfo-NHS-LC-Biotin should I use to label antibodies and other proteins?
The molar excess to use depends on the concentration of the ligand to be biotinylated. Although the literature contains references that use between 2-50 molar excess of biotin, we recommend the following: 10-fold molar excess for 10 mg/ml solution of protein; 25-fold molar excess for 2 mg/ml solution of protein.

Why can't I perform biotinylation reactions in Tris buffer with Sulfo-NHS-LC-Biotin?
Tris is a primary amine, which will directly compete with protein primary amines for reaction with the NHS-ester activated biotin reagent. However, once a protein is labeled and desalted, Tris buffer can be used for storage or assay buffers, as appropriate for the protein and application.

Can Sulfo-NHS-LC-Biotin be used in cell surface biotinylation?
Yes. Because this molecule dissolves readily in polar solutions and is itself charged by the sodium sulfoxide group on the succinimidyl ring, it cannot permeate the cell wall or membrane of an intact cell. As long as the cell is intact, only the primary amines on the surface will be biotinylated.

Can I make a stock solution to use later?
No. NHS esters hydrolyze very quickly. The container must be brought to room temperature (RT) prior to opening, then the amount of biotinylation reagent weighed, dissolved and used immediately.

Are similar biotinylation reagents with different spacer arm lengths available?
Yes. We offer a wide selection of amine-reactive biotinylation reagents. These include reagents with longer spacer arms, polyethylene glycol spacer arms and cleavable spacer arms. Several convenient biotinylation kits are also available.

Is there a cleavable analog of Sulfo-NHS-LC-Biotin?
Yes. Sulfo-NHS-SS-Biotin (Product # 21331) has a disulfide bond in the spacer arm that can be cleaved with DTT or other reducing agents, thereby releasing the biotin tag from the previously labeled protein.

How can I determine the amount of biotinylation on my ligand?
An assay with HABA dye (Product # 28010 or Kit # 28005) can be used to determine the molarity of biotin in solution.

 

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