EZ-Link™ Micro Sulfo-NHS-LC-Biotinylation Kit - FAQs

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.

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All four Micro Biotinylation Kits will label proteins for use in many applications. Consider the following guidelines for choosing among the kits:

• Choose the Sulfo-NHS-Biotinylation Kit (Cat. No. 21925) when it is important to minimize the molecular mass contributed by the label.
• Choose the Sulfo-NHS-SS-Biotinylation Kit (Cat. No. 21945) only when the downstream application require cleaving the biotin tag with a reducing agent.
• Choose the NHS-PEG4-Biotinylation Kit (Cat. No. 21955) to maximize protein solubility and prevent protein aggregation during storage.
• Choose the Sulfo-NHS-LC-Biotinylation Kit (Cat. No. 21935) for routine purposes when a feature of one of the other reagents is not required. The longer spacer arm offers the possibility of optimizing detection experiments where steric hindrance of biotin binding is an important factor.

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The entire procedure can be completed in less than 3 hours.

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Any molecule containing a primary amine can be labeled. Any protein that has been labeled using the standard biotinylation kits, including antibodies and peptides, also can be labeled with the micro kits.

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Only the sulfo-NHS-SS-Biotin is cleavable because it has a disulfide bond in its spacer arm, enabling labeled proteins to be cleaved from the biotin group by treatment with reducing agents.

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The reagents in the Micro kits are amine reactive. NHS esters react with a-amines at the N-termini and the epsilon-amines of lysine side chains at pH 7-9 to form stable amide bonds and the release of NHS.

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Yes, the four Micro kits contain water-soluble biotinylation reagents; three kits contain sulfo-NHS reagents and one kit contains a polyethylene glycol (PEG) reagent. The sulfo-NHS ester imparts water solubility and is the reaction leaving group. The hydrophilic PEG spacer arm imparts water solubility that is maintained after conjugation.

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There are four Micro Biotinylation Kits as shown below:

• EZ-Link Micro Sulfo-NHS-Biotinylation Kit, Cat. No. 21925
• EZ-Link Micro Sulfo-NHS-LC-Biotinylation Kit, Cat. No. 21935
• EZ-Link Micro Sulfo-NHS-SS- Biotinylation Kit, Cat. No. 21945
• EZ-Link Micro NHS-PEG4-Biotinylation Kit, Cat. No. 21955


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The HABA assay requires a large volume of biotinylated sample. Using the HABA assay with the Micro Biotinylation kits consumes too much of the sample. An alternative verification method is to perform a dot blot of the biotinylated protein using streptavidin-HRP or other labeled avidin product.

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The standard biotinylation kits contain components for labeling 1-10 mg of protein, and the Micro Biotinylation Kits are structured for labeling 50-200 µg of protein in 200-700 mL. The Micro kits contain the biotinylation reagents in small, individual packages and do not include components to perform the HABA assay.

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EZ-Link Micro Sulfo-NHS-LC-Biotinylation Kit is covered under our general 1-year warranty and is guaranteed to be fully functional for 12 months from the date of shipment, if stored as recommended. Please see section 8.1 of our Terms & Conditions of Sale (https://www.thermofisher.com/content/dam/LifeTech/Documents/PDFs/Terms-and-Conditions-of-Sale.pdf) for more details.

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Sometimes. The biotin has to insert 9 angstrom into the pocket of avidin, streptavidin or NeutrAvidin to bind. If steric hindrance may be a problem, using a biotinylation reagent with a longer spacer arm (Sulfo-NHS-LC Biotin or Sulfo-NHS-LC-LC Biotin) may result in greater purification or deteciton.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

The sodium sulfonate group lends water solubility to the reagent.
Note: The actual biotin moiety that is added is identical between NHS and Sulfo-NHS biotins that vary only in the addition of the sodium sulfonate on the leaving group. Researchers preferring a more water soluble biotin be added to their molecule should consider a PEGylated version of biotin.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.