Structure of EZ-Link Biotin Hydrazide Labeling Reagent .

Structure of EZ-Link Biotin-LC-Hydrazide Labeling Reagent

Biotin Hydrazide labeling reagents enable simple and efficient biotin labeling of antibodies and other glycoproteins. Mild oxidation of antibodies with sodium periodate produces reactive aldehydes on the carbohydrate moieties of the Fc portion that can be modified by hydrazides. This approach is advantageous for labeling antibodies because biotinylation occurs only at the sites of glycosylation, which are primarily in the Fc region of the antibody, far from the antigen binding site. Antibodies biotinylated in this manner thus retain complete immunological reactivity¹. This method is ideal for polyclonal antibodies because they are heavily glycosylated. Success with monoclonal antibodies depends on the extent of glycosylation for a particular antibody.

Biotin Hydrazide and Biotin-LC-Hydrazide bind to oxidized carbohydrates through the hydrazide group (–NH-NH₂), forming a hydrazone linkage. Oxidation of glycoproteins generates reactive aldehydes that react specifically with hydrazide groups. Sialic acid residues on glycoproteins can be specifically oxidized with sodium periodate (NaIO₄) by using 1 mM periodate and a temperature of 0°C. Sialic acid residues also can be biotinylated with hydrazide derivatives by pretreatment with neuraminidase to generate galactose groups. The galactose and
N-acetylgalactosamine residues on whole cells can be selectively biotinylated by further treatment with galactose oxidase, which will convert the primary hydroxyl groups on these sugars to their corresponding aldehydes.

Reaction of Biotin Hydrazide with an oxidized glycoprotein.

Additionally, these reagents can be reacted with carboxyl groups on aspartate and glutamate residues or on the carboxy terminus. The reaction is mediated by EDC, which activates carboxyl groups to bind to the -NH₂ group on the biotinylation reagent, forming an amide linkage. Using EDC results in some polymerization of the peptide or protein if the molecule contains both carboxyls and primary amines. Decreasing the amount of EDC or increasing the amount of biotin reagent in the reaction can minimize polymerization.

Biotin Hydrazide Labeling Reagents Highlights

• Label glycoproteins with biotin to facilitate immobilization, purification or detection
• Attaches a biotin label to antibodies through carbohydrate modifications in the Fc region
• Biotin Hydrazides react with oxidized carbohydrates (aldehydes) at pH 4-6 to attach biotin through stable hydrazone bonds
• Biotin hydrazides can be used to specifically label cell surface glycoproteins²

Biotin Hydrazide and Biotin-LC-Hydrazide Reagent Specifications

Pierce manufactures biotin labeling reagents to the highest specifications to produce the most specific biotin labeled proteins, ensure the integrity of your data and to provide you with the highest degree of consistency.
Each lot of Biotin Hydrazide and Biotin-LC-Hydrazide labeling reagents are tested to meet our purity and solubility specifications.

Considerations for use of Biotin Hydrazides

• Avoid Tris or other primary amine-containing buffers in the oxidation and biotinylation steps as these buffers react with aldehydes and will quench the reaction with hydrazides.
• Biotin Hydrazide and Biotin-LC-Hydrazide are soluble in aqueous buffers up to approximately 5 mM. They can also be dissolved in DMSO at concentrations up to 50 mM, then diluted into aqueous reaction mixtures; however, Biotin- Hydrazide and Biotin-LC-Hydrazide are poorly soluble in DMF.
• Temperature, pH of oxidation and periodate concentration affect the reaction with hydrazides. Also, because glycosylation varies with each protein, optimal conditions must be determined for each glycoprotein. Each glycoprotein preparation will have an optimum pH for oxidation and an optimum pH for the hydrazide biotinylation.

Related products
Amine-reactive Biotinylation Reagents
Sulfhydryl-reactive Biotinylation Reagents (Maleimide- and Iodoacetyl-containing )
Carboxyl-reactive Biotinylation Reagents
Carbohydrate-reactive Biotinylation Reagents
Specialty Biotinylation Reagents (Includes photoreactive reagents for nonspecific protein labeling)
Biotin Quantitation (EZ Biotin Quantitation Kit and HABA Dye)
Immobilized Avidin, Streptavidin, NeutrAvidin and Monomeric Avidin

Other useful links
Biotinylation Reagents Selection Guide
Biotin labeling and other reactions in Slide-A-Lyzer Dialysis Cassettes