Biotin Protein Labeling Kit Protocol & Troubleshooting

Product No. 11418165001


Alkaline conditions (pH 10-10.5)

Conjugation at pH 10 - 10.5 has not been tested. At higher pH, hydrolysis of the ester could occur, and a pH shift to 10-11 will disrupt the ester.

When alkaline conditions are required for protein solubility reasons, try labeling at incremental increases in pH starting from the pH optimum of the ester, to see if a balance of labeling of your specific protein with a minimal amount of ester hydrolysis can be achieved.


It is not known whether preservatives impair the labeling reaction. No direct interference is expected especially not from sodium azide. For best results, follow the above described reaction conditions, especially the recommendations for composition of the reaction buffer, as described in the Instruction Manual:

The reaction buffer must be amine-free.
The pH of the reaction buffer is also critical. Roche recommends using PBS with a pH of 7.4. Reaction kinetics will increase with a more alkaline pH while being decreased with a more acidic pH.

When in doubt concerning the composition of the reaction buffer, Roche recommends dialyzing the antibody and performing the reaction in PBS.

Small Sample Amounts

Sephadex G-25 columns provided with the kit allow purification of protein amounts down to 100 μg. For less than 100 μg protein per reaction, use ultrafiltration devices, such as Microcon from Amicon; processing volume 0.005-0.5 ml or microdialysis.

When using less than 1 mg protein per reaction, use the molar ratios as given in the standard protocols of the Instruction Manual. For protein concentrations lower than 1 mg/ml, use higher amounts of Biotin-NHS.

Labeling of Small Peptides (1.3 kD)

Small proteins or peptides in this molecular weight range have not been tested.
In general, there could be problems separating nonreacted biotin-7-NHS molecules from the labeled protein using the Sephadex G-25 column, because the molecular weight of biotin-7-NHS is approximately 700 Da.

Instead of using the Sephadex G-25 column, add a glycine buffer (1 mol/l) for binding nonreacted biotin-7-NHS using an excess of free amino groups.

It is also possible to replace biotin-7-NHS with D-biotin-NHS ester (mol. weight 341 Da) when incorporating biotin residues into amino acids, peptides or proteins. This approach provides a better size discrimination between nonreacted molecules and labeled peptide.

Regeneration of columns

Sephadex G-25 columns can be easily regenerated after use: Simply rinse the column with 30ml PBS solution (6 x 5ml) and it will be ready for use again. If the column is not re-used immediately, close it with stopper and cap and store at +2 to +8 °C. Columns can be re-generated up to 5 times.

Inactivation of NHS-ester

The biotin-NHS-ester is stable under acidic conditions, but labile under basic conditions; note that a shift to pH 10-11 probably destroys the ester, and can also disrupt the labeled protein.

Another option to inactivate the biotin-NHS-ester is to add another amine, e.g., ethanolamin; this would stop the labeling reaction by competition. Add equimolar amounts (or slightly more) of ethanolamine.

Although spontaneous hydrolysis of biotin-NHS-ester occurs, do not depend on this occurring. The biotin-NHS-ester is relatively stable under the recommended labeling conditions, and the incubation time of 2 hours is not sufficient. To remove the ester by hydrolysis, continuously stir the labeling reaction for at least 1 to 2 days!

In summary: To get separate the ester, Roche recommends using chromatography or dialysis.


Low labeling rate

  • pH of conjugation mix was not well adjusted: The pH should be between pH 7 to 9.
  • The protein to be coupled was in a buffer containing primary amino groups or sugars: Dialyze your protein against PBS.
  • Biotin-7-NHS solution was not freshly prepared: Use only freshly prepared Biotin-7-NHS.
  • Protein concentration was too low: Optimal labeling rates are achieved with protein concentrations from 5 to 15 mg/ml.
  • Proposed stoichiometry of reaction mix is not well suited for your protein: Try other protein to Biotin-7-NHS ratios.

Turbid eluate

  • Centrifuge the solution at high speed prior to use.


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