b'General western blotting (continued)ObservationCauseSolution Contamination ofPrepare fresh buffers and filter them before use.equipment or materials Use only clean and contaminant-free electrophoresis equipment, blotting equipment, and incubation trays.High background Reduce the length of time the blot is exposed to film.(continued) Reduce the concentration of the substrate.Signal fromShorten incubation time of membrane with substrate.chemiluminescentCompletely remove substrate after incubation period.substrate too strongDecrease the concentration of antibodies, particularly HRP- and AP-conjugated antibodies.After transfer, stain the gel with a total protein stain to determine transfer efficiency, such as Ponceau S or the Thermo Scientific Pierce Reversible Protein Stain Kit (Cat. No. 24585 or 24580).Ensure sufficient contact between the gel and membrane during transfer by using a gel roller across the transfer stack.Ensure that the stack is placed in the transfer apparatus in the proper orientation such that proteins will migrate onto the membrane.Wet and activate the membrane according to the Incomplete ormanufacturers instructions.inefficient transfer Use a positive control, such as prestained molecular weight markers, to quickly assess whether transfer has occurred.Use molecular weight markers compatible with a western imaging substrate, such as the Invitrogen iBright Prestained Protein Ladder (Cat. No. LC5615) or Invitrogen MagicMark XP Western Protein Standard (Cat. No. LC5602), as a positive control.Increase transfer time and/or voltage.Make sure sample preparation conditions have not destroyed the antigenicity of the sample. (Some proteins cannot be run under reducing conditions.)For low molecular weight (MW) targets, add 20% methanol to the transfer buffer to help binding and prevent proteins from passing through the membrane.Insufficient bindingReduce transfer time. Low MW proteins may pass through the membrane.to membrane For high MW targets, add 0.010.05% SDS to the transfer buffer to facilitate the movement of proteins from the gel to the membrane.Weak signal orChange membrane type (nitrocellulose vs. PVDF).no signal Change to membrane with smaller pore size.Antibody concentrationIncrease antibody concentrations. Antibody may have poor affinity for the too low target protein.Antibody may have lost activity. Perform a dot blot to determine activity.Insufficient target proteinLoad more protein onto the gel.presentDecrease concentration of protein in blocking buffer.Protein masked byTry a different blocking buffer. Use our blocking buffer selection guide at blocking buffer thermofisher.com/blockingbuffers to find the most compatible blocking buffer for your experiment.Buffer containsSodium azide inhibits HRP. Do not use it with HRP-conjugated antibodies.sodium azideIncrease incubation time of membrane with substrate.Signal fromIncrease film exposure time.chemiluminescentEnsure that the substrate is not expired.substrate too weak When you have minimal protein, use Thermo Scientific SuperSignal West Atto Ultimate Sensitivity Substrate (Cat. No. A38555) to maximize your western blot signals.Membrane has beenAvoid repeated stripping of the same membrane.stripped and reprobed Shorten incubation time in stripping buffer to prevent loss of target protein.Digestion of proteinBlocking buffer may contain a substance that has proteolytic activity (e.g., on membrane gelatin); try changing the blocking buffer to a purified blocking protein such as casein.Protein degradation fromPrepare new blot.prolonged blot storage81'