b'Fluorescent western blotting (continued)ObservationCauseSolution Handle the membrane using clean forceps and clean incubation trays or dishes.High backgroundDetermine the best blocking buffer for your applicationprimary antibodies due to membranewill react differently in different blocking buffers. Blocking buffers like normal contamination animal sera or milk may result in cross-reactivity. Use our blocking buffer selection guide at thermofisher.com/blockingbuffers to find the most compatible blocking buffer for your experiment.Artifacts fromLoad less of the molecular weight marker onto the gel. overloading the protein marker or ladderNonoptimal wash orUse a wash buffer with 0.10.2% Tween 20 detergent.diluent solutions Prepare the secondary antibody dilution with 0.05% Tween 20 detergent.Increase the number or duration of wash steps.High backgroundOptimize the secondary antibody dilution depending on the dye being used, Background issues from an excess offollowing the vendor-recommended dilution and adapting accordingly.(high, uneven, orsecondary antibodyspeckled) Blotchy or unevenEnsure good coverage of the whole blot during all incubation steps.background due to the membrane drying out Ensure consistent agitation during every incubation step.Incorrect choiceThe nature of the membrane can affect the background; for example, of membrane PVDF membranes can autofluoresce and cause high background, so use low-fluorescence PVDF membranes.Use clean forceps to handle the membrane, and avoid directly touching the membrane; particulates and contaminants from unclean tools may fluoresce.Speckles andUse clean incubation trays or dishesrinsing with methanol followed by fingerprints on thewater will help dissolve residual dried dyes from previous uses. membrane Clean transfer devices and dusty consumables if using a wet transfer method, as they can introduce speckles.Clean the imager surface with ethanol to remove dust, lint, and residue before capturing the image.83'