High Background in Rapid Immunodetection Protocol

Symptom

When using rapid immunodetection methods, membrane shows elevated background specific to fast protocols. Issue not present with standard overnight protocols.

1. 1 Membrane wets out prematurely during rapid immunodetection due to high Tween-20 concentration (>0.04%)
2. 2 Vigorous agitation during rapid incubations causes membrane wetting
3. 3 Residual SDS from gel electrophoresis carried over to membrane
4. 4 Membrane pre-wetted in methanol before starting rapid protocol
5. 5 Membrane not completely dry before beginning rapid immunodetection

1. 1 Reduce Tween-20 concentration in antibody diluent to <0.04%
2. 2 Use gentler agitation during rapid incubation steps
3. 3 Rinse blot thoroughly in Milli-Q water after electrotransfer to remove residual SDS from gel
4. 4 Do not pre-wet membrane in methanol before rapid immunodetection protocol
5. 5 Ensure membrane is completely dry before starting rapid detection procedure; air dry or use blotting paper

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