Determination of biotin on a protein by quantitative sodium dodecyl sulfate-capillary gel electrophoresis of monomeric avidin.
Sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) is performed to quantify monomeric avidin and biotin on a protein. Under non-reducing SDS-CGE conditions, avidin migrates as monomers exhibiting apparent molecular mass 17,000. In the presence of a biotin-protein conjugate, monomeric avidin binds the conjugate and forms a larger complex that migrates later in the separation. The difference between the remaining monomeric avidin and the initial amount is the portion of monomeric avidin bound to the conjugate. Accordingly, the number of biotin on the protein can be calculated. The assay is linearly responsive to increasing biotin loading in a biotinylation reaction of a protein. Accuracy of the assay is also demonstrated by good sample dilution recovery. Excellent quantitative reproducibility < 2% (relative standard deviation) is obtained for both intra- and inter-day measurements. Main advantages of the method include the use of monomeric avidin that minimizes steric hindrance to capture biotin on a protein and assay automation on a capillary electrophoresis apparatus.
Lee
Huey G
HG
Analytical Research Laboratory, Bayer Diagnostics, 511 Benedict Avenue, Tarrytown, NY 10191, USA. huey.lee.b@bayer.com
Fritsche
Edward
E
D001360:Avidin / Q000302:isolation & purification*; D001710:Biotin / Q000032:analysis*; D019075:Electrophoresis, Capillary / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D015203:Reproducibility of Results; D012967:Sodium Dodecyl Sulfate
D011506:Proteins; D001360:Avidin; D012967:Sodium Dodecyl Sulfate; D001710:Biotin