Labeling Chemistry Support—Getting Started

• The molecule to be labeled:
  • Must have a suitable site for labeling, e.g., primary amine, thiol, carboxyl, phosphate, etc.
  • Must be soluble and stable in the reaction solution 
  • Must be amenable to some form of post-reaction purification to separate unbound label from labeled molecule. Post-reaction purification may be by the use of size exclusion chromatography, dialysis, HPLC, electrophoresis, or other methods.

• The site of attachment must be accessible to the reactive label.
• The desired reactive site on the molecule must not have competing sites. For example, if using carbodiimide-activation (e.g., using EDAC, CDC) of carboxyls to attach an amine-terminated label, the molecule must not have any accessible phosphates, as phosphates will be activated as well and become labeled. 

You must know something of the structure of your molecule, peptide, or protein to know what sites are suitable for labeling and that the intended site for labeling is accessible to the reactive labeling reagent. Avoid covalently modifying the active sites of enzymes or other strategically functional areas of the molecule. For example, if the carboxyl terminus of a peptide is required for binding or other function, avoid conjugating a dye to the carboxyl group or anywhere near the C-terminus. 

No. For large molecules that may have more than one of the same reactive site for labeling, e.g., lysines on the surface of a protein, labeling with the amine-reactive reagent is ‘shotgun’ or random. It is not possible to control what sites will be conjugated, and the final product will be a mixed population of labeled protein with the label at different sites throughout the surface of the individual protein. 

• First, select a fluorophore that matches the light source and filters/channels available on your instrument to guarantee a suitable level of excitation and emission for adequate detection.
• Second, know something about the properties of your molecule. Is your molecule hydrophilic, hydrophobic, or overall cationic or anionic? You may wish to label a hydrophobic molecule with a hydrophobic dye such as our BODIPY™ dyes to retain the overall hydrophobic properties of the molecule. If you have a peptide or other molecule that is inherently cationic, you may wish to attach a cationic rhodamine dye. If the molecule is largely anionic, use a dye that is anionic such as fluorescein or some of the Alexa Fluor™ dyes. 
• Third, determine the site and extent of labeling that is suitable for your molecule. For example, a single quantum dot (Qdot™ probe) attached to the the Fc portion of a whole antibody (SiteClick™ kits) may be a better option than organic dyes attached everywhere on the surface of the antibody. For antibodies that may have lysines on or near the antigen binding site of the antibody, labeling with an amine-reactive dye may render the antibody unusable. 

We highly recommend that you determine the DOL. DOL determination is achieved by absorbance readings at either 260 nm (for nucleic acids) or 280 nm (for proteins) plus the absorbance maximum for the fluorophore. It is only by an absorbance reading that you may determine if the labeling reaction was successful and, if subsequent labeling of the same molecule is to be done in the future, to establish the best level of labeling for that molecule.

Fluorescence detection cannot tell you if the molecule is unlabeled, under-labeled, or overlabeled— all of these labeling scenarios may provide the same level of little or no fluorescence. With some dyes, overlabeling may result in dye-dye quenching, resulting in no detectable fluorescence, but the dye will be detected using absorbance. Also, visual inspection of the color of the solution of the labeled molecule cannot determine if it was suitably labeled. 

A DOL measurement may be performed for biotinylated-molecules using either the HABA assay or similar assays (see Cat. No. B30751 FluoReporter™ Biotin Quantitation Assay Kit). 

Yes. Depending upon the reaction chemistry, labeling may be carried out in organic solvents such as DMSO, DMF, acetonitrile, alcohol, or other solvents. Useful information may be found in the text Bioconjugate Techniques (Greg T. Hermanson, Academic Press) or at Labeling Small Peptides with Amine-Reactive Dyes in Organic Solvents.

Reasons for the loss of signal from a fluorophore-conjugated molecule are varied, depending if the loss was due to the properties of the molecule or due to the fluorophore attached to the molecule. Poor signal may be due to poor binding or a higher on/off rate relative to the unlabeled molecule. Proteins or other molecules may be digested by proteases and the subsequent product comprising small, fluorophore-conjugated peptides or amino acids may become too diffuse for detection or wash away. Some fluorophore-conjugated molecules may be actively effluxed by live cells to a much greater extent than the unlabeled molecule. 

Fluorophores attached to the probe of interest may become photobleached, quenched, or degraded. The presence of colorimetric dyes (e.g., phenol red, trypan blue), xylene, or other agents may transiently quench some fluorophores; once removed, the fluorophores are still fluorescent. Fluorophores may become irreversibly degraded by photobleaching, oxidation, reduction, or by other means. Avoid exposure to light during storage, the extremes of pH, strong oxidizing or reducing agents, heavy metals, and, for fluorescent proteins such as GFP or the phycobiliproteins (R-PE, APC), avoid anything that may promote digestion (i.e., by proteases), unfolding, or denaturation.

Each kit contains 3 vials of solid sodium bicarbonate (Zip buffer reagent, Component A), 3 vials of the Alexa Fluor reactive dye (Component B), and a Quick Reference Card. Sufficient reagents are provided to label 100 µg of antibody per reaction (100 µL of a 1 mg/mL antibody solution), 3 reactions per kit.

The 1 mg/mL concentration of the antibody (100 µg of antibody in a 100 µL volume) allows for optimal reaction kinetics. A more dilute solution of antibody would exhibit suboptimal labeling due to a greater amount of reactive dye lost to spontaneous hydrolysis; the reactive group on the dyes is labile in aqueous solutions.

Yes, the BSA and any component with a primary amine (such as the Tris buffer) would react with the amine-reactive Alexa Fluor dyes used in these labeling kits, resulting in less optimal labeling of the antibody. For small volume purification of the antibody, you may use either Nab Protein A/G Spin Kit, 0.2 mL (Cat No. 89950), Protein A/G Magnetic Agarose Beads (Cat No. 78609), or Melon Gel IgG Spin Purification Kit (Cat No. 45206).

This is because we have optimized the amount of dye to label 100 µg of antibody per reaction and after the incubation time of 15 mins, most of the reactive dye is either bound or unreactive due to spontaneous hydrolysis, leaving little or no reactive dye to label your sample.

No, it is intended for immediate use. If you wish to store your labeled antibody after the reaction, it should undergo a post-reaction clean-up using either size exclusion resin (such as Bio-Rad BioGel P-30) or Dye Removal Columns (Cat No. 22858).