Biotinylation of Proteins

Dominik Haudenschild, November 12, 2002 at UCD

 

Using EZ-LINK Sulfo-NHS-LC-Biotinylation Kit (Pierce 21430)

 

  1. Use 2-10mg protein in 1ml PBS
    Calculate mmoles of protein  =  (mg protein)Ö (Mol. Weight)

  2. Add 20-fold molar excess of Sulfo-NHS-LC-Biotin

  3. Incubate 2 hr on ice or 30min RT

  4. Equilibrate 10ml desalting column with 30ml PBS

  5. Desalt Sample, collect 1ml fractions, check OD280

  6. Store at Ð20¡ (or temporarily at 4¡) in 0.1% Sodium Azide

 

Calculations
                  (mmoles protein) x 20 = mmoles biotin reagent to add

     (mmoles biotin reagent) x 556 = mg biotin reagent to add

                                    Antibodies  = 150,000 Molecular Weight