b'Fluorescent western blottingObservationCauseSolution Poor antibody specificityEvaluate additional primary antibodies.for the target of interest Use only primary antibodies validated* for western blots.Sample degradation due to overheating or protease activity results in target Poor sample integrity breakdown and low target recognition by the antibody. For example, do not boil SDS-PAGE samples in SDS sample buffer, but rather heat them at 70C for 10 minutes to avoid proteolysis.Choose primary antibodies raised in distantly related host species.Nonspecific orAntibody cross-reactivityUse highly cross-adsorbed secondary antibodies.diffuse bands in multiplex detection Reduce the amount of the secondary antibody used, to remain within the optimal performance range.Fluorescent bleed- Avoid spectrally close conjugates, especially when the signal is very strong.through from anotherEnsure that your fluorescent dyes can be distinctly detected on your channel whenimaging instrument.multiplexing (appearanceIf available, use the autoexposure feature on your imaging instrument to of an unexpected band) determine the optimal exposure time(s) for each channel.Increase primary antibody concentration.Ensure that the primary antibody has a good titer and is specific for the target protein.Insufficient amount ofFor a low-abundance target in a cell or tissue lysate, increase the amount of primary antibody primary antibody or the amount of sample loaded on the gel.Extend the incubation time to overnight at 4C, or 36 hours at room temperature.Try using an antibody enhancer.Ensure the antibody was stored appropriately.Lost activity of antibody Check the expiration date of the antibody.Avoid multiple uses of prediluted antibodies.Imaging exposure time isIncrease exposure time.too short If available, use the autoexposure feature on your imaging instrument to determine the optimal exposure time(s).Incorrect instrumentEnsure the correct excitation and emission ranges are selected for the Weak or no signal settings intended fluorophore.Use of detergent Too much detergent or the nature of the detergent can result in washing away the signaldecrease or eliminate detergent.Some blocking solutions can mask the blot and reduce the availability of the Blocking buffertarget protein to the antibody, especially if the blocking step is 1 hour.blocks target protein Dilute the primary antibody in wash buffer.Evaluate another blocking buffer.Too much lysate can overcrowd your specific target and reduce the Quantity of sampleantibody sensitivity.loaded on the gel Too little lysate leads to insufficient availability of the target of interest.Perform serial dilutions of the lysate or sample to determine the optimal amount of protein to load.Poor transfer of protein,Check transfer conditions to confirm protein transfer.or loss of the protein after transfer Reoptimization may be required when probing for a new protein.* The use or any variation of the word validation refers only to research use antibodies that were subject to functional testing to confirm that the antibody can be used with the research techniques indicated. It does not ensure that the product(s) was validated for clinical or diagnostic uses.82'