High Background Troubleshooting

My conjugate gives a high background.

 

Background problems may arise in some applications if your conjugate contains too much free xlAPC. You can reduce the amount of free xlAPC in the conjugate by lowering the ratio of SMCC-xlAPC to antibody in the conjugation reaction. Take care in doing this because the change may also result in larger conjugate aggregates which can also give rise to background problems in some applications.

Free xlAPC may be removed by purifying the conjugate with a size-exclusion chromatography column. We have found that Bio-Gel A-0.5m (Bio-Rad) works well for antibody-xlAPC conjugates. For optimal results the volume of conjugate applied to the column should not exceed 5% of the column volume.