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l Improper washing
l Substrate was contaminated
l Non-specific binding of antibody
l Plate are not be sealing incompletely
l Incorrect incubation temperature
l Substrate exposed to light prior to use
l Contaminated wash buffer
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l Increasing duration of soaking steps
l Replace. Substrate should be clean and avoid crossed contamination by using the sealer
l Replace another purified antibody or blocking buffer
l Make sure to follow the instruction strictly
l Incubate at room temperature
l Keep substrate in a dark place
l Use a clean buffers and sterile filter
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l Improper washing
l Incorrect incubation temperature
l Antibody are not enough
l Reagent are contaminated
l Pipette are not clean
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l Increasing duration of soaking steps
l Incubate at room temperature
l Increase the concentration of the antibody
l Use new one
l Pipette should be clean
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l Reagent are contaminated
l Sample prepared incorrectly
l Antibody are not enough
l Wash buffer contains sodium azide
l HRP was not added
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l Use new one
l Make sure the sample workable/dilution
l Increase the antibody concentration
l Use a new wash buffer and avoid sodium azide in it
l Add HRP according to the instruction
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l Imprecise/ inaccurate pipetting
l Incomplete washing of the wells
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l Check/ calibrate pipettes
l Make sure wells are washed adequately by filling the wells with wash buffer and all residual antibody solutions crossed well before washing.
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