Rigorous purification of the antibody/DNA conjugate is crucial for the sensitivity of the assay. Different methods to remove non-conjugated antibody and free DNA can be used. We recommend first removing free antibody with anion-exchange chromatography followed by gel filtration to separate the free DNA from the conjugate.
Ion-exchange column: Resource® Q 1 ml
Buffers: 10 mM Tris pH 8.0 in 0 M NaCl and in
1.5 M NaCl for gradient elution.
Identify the fractions containing free DNA and antibody/DNA conjugate by absorption spectroscopy. Pool and concentrate these fractions using a Centricon YM-100 (Millipore), or similar, to a volume (typically about 0.5 ml)
suitable for the gel filtration column to be used.
Gel filtration column: Superdex 200 HR10/30
Buffer: 50 mM Tris, 1 mM EDTA, 0.15 M NaCl, pH 8.0.
Collect eluate with fraction collector Fractions to use in real-time immuno-PCR are best identified in test runs. The amount to use in the test runs can be estimated by regular real-time PCR. Fractions containing active conjugate are pooled and stored either in refrigerator or freezer. About 5 mg ml-1 of BSA can be added to stabilize the solution. Although it may depend on the antibodies, we have found stock conjugate solutions to be stable upon storage at +4°C or -20°C for at least 1 year.
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