b'Troubleshooting tipsGeneral western blottingObservationCauseSolution Antibody concentrationReduce concentrations of antibodies, particularly of primary antibody.too highToo much protein loadedReduce the amount of sample loaded on gel.on gelNonspecific or diffuseReduce the length of time the blot is exposed to film.bands Signal fromReduce the concentration of the substrate.chemiluminescentShorten incubation time of membrane with substrate.substrate too strong Completely remove substrate after incubation period.Decrease the concentration of antibodies, particularly HRP- and AP-conjugated antibodies.Ensure sufficient contact between the gel and membrane during transfer by using a gel roller across the transfer stack.Partially developedIncomplete transfer Wet and activate membrane according to manufacturers instructions.areas or blank areasAlways wear clean gloves or use forceps when handling membrane.Antibody concentrationDecrease concentration of primary and/or secondary antibody.too highDo not use milk with avidinbiotin system. Milk contains biotin, which will result in high background. When probing for phosphoproteins, avoid phosphate-based buffers like PBS and phosphoprotein-containing blockers like milk or casein. Instead, block with BSA in Tris-buffered saline.Test for cross-reactivity in blocking buffer by blocking a clean piece Incompatibleof membrane, incubating with antibodies, and then detecting with the blocking buffer substrate of choice.When using an alkaline phosphatase (AP) conjugate, a blocking buffer in Tris-buffered saline (TBS) should be selected because phosphate-buffered saline (PBS) interferes with AP activity.Try a different blocking buffer. Use our blocking buffer selection guide at thermofisher.com/blockingbuffers to find the most compatible blocking buffer for your experiment.Increase the concentration of protein in the blocking buffer.Optimize blocking time and/or temperature. Block for at least 1 hour at room temperature (RT) or overnight at 4C.Adding Tween 20 detergent to the blocking buffer can help minimize High background background. However, too much detergent can interfere with antibody binding. A final concentration of 0.05% often works well. For ease of use, choose a blocking buffer that already contains 0.05% Tween 20 Insufficient blocking ofdetergent, such as Thermo Scientific StartingBlock T20 Blocking nonspecific sites Buffer (Cat. No. 37543 or 37539) or SuperBlock T20 Blocking Buffer (Cat. No. 37536 or 37516).Prepare antibody dilutions in a blocking buffer that contains 0.05% Tween 20 detergent.Use Thermo Scientific SuperSignal Western Blot Enhancer (Cat. No. 46640) to reduce background and enhance detection oflow-abundance and weakly immunoreactive target proteins.Increase the number of washes and/or the volume of buffer used.Insufficient washing Add Tween 20 detergent to the wash buffer to a final concentration of 0.05%. If the concentration of Tween 20 detergent is too high, it can strip proteins off the membrane.Wet and activate membrane according to manufacturers instructions.Always wear clean gloves or use forceps when handling membrane.Membrane handledCover the membrane with liquid at all times to prevent drying.improperly Use agitation during all incubations.Handle membrane carefullydamage to the membrane can cause nonspecific binding.80'