Biotin NHS Ester ((+) Biotin N-hydroxysuccinimide ester) |CAS 35013-72-0| HPT1802

Biotin NHS Ester Biotinylation Reagent

For Peptide and Oligonucleotide Labeling

Biotin-NHS ester is an amine reactive biotinylation reagent employed to label primary amines on peptides and oligonucleotides.

Reaction scheme for conjugation to primary amines. Biotin-NHS ester is efficiently conjugated to epsilon amines on lysine residues, other primary amines, and thiol groups.

Protocol

Biotinylation protocol to label cellular proteins in-situ with biotin (for one 10 cm cell culture dish of confluent cells).  Adjust volumes accordingly for other size plate of cells. 

Required Reagents and Equipment

1. PBS pH 7.6
2. DMSO
3. Low speed centrifuge used for cell culture
4. Rocker table

All biotinylation steps should be performed at room temperature.

1. Gently wash cells in PBS pH 7.6 three times for remove media and serum proteins.
2. Cover cells in 4 mls of PBS pH7.6.
3. Suspend Biotin-NHS ester in DMSO (use 250 microliters DMSO for 0.5 mg Biotin-NHS ester).
4. For a 10 cm cell culture dish, add 100 microliters of the Biotin-NHS ester DMSO solution mixed above to the plate of cells with PBS pH 7.6.  
5. Use a rocker table, and mix gently for 30 min. Cells will detach during this step.
6. Add 2 ml 100 mM glycine in PBS pH 7.6 to quench the reaction.
7. Gently collect cells with the glycine-PBS solution and centrifuge at 3000 rpm in a cell culture centrifuge. 
8. Wash cells 3 more times in the 100 mM glycine-PBS solution.  Remove all glycine-PBS
9. Extract proteins using a cell lysis buffer. 

The lysate can be stored at -20^oC or -80^oC for later procedures, including binding to streptavidin linked solid support. 

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