Spots lack sharp borders and appear fuzzy or poorly defined, making automated or manual counting difficult. The reader has trouble distinguishing individual spots.
Common Causes
1Membrane not pretreated with ethanol before coating
2Plate movement during cell incubation causing cells to migrate and create multiple spots
3Coating antibody concentration too low (especially for fluorescence detection)
4Residual ethanol trapped between membrane and plate bottom due to inadequate washing
5Cells distributed unevenly creating irregular cell density
Solutions
1Pretreat membrane with 35% ethanol; wash thoroughly with PBS 3× to remove all ethanol
2Do not move, stack, or tap plates during cell incubation; use dedicated incubator
3Increase antibody concentrations for both coating and detection steps
4Ensure complete washing after ethanol pretreatment to remove trapped ethanol
5Mix cells gently to obtain homogenous suspension before pipetting into wells