Reduce the length of time the blot is exposed to film. Signal from chemiluminescent substrate too strong Use only clean and contaminant-free electrophoresis equipment, blotting equipment, and incubation trays. Prepare fresh buffers and filter them before use. Handle membrane carefully-damage to the membrane can cause nonspecific binding. Wet and activate membrane according to manufacturer’s instructions.Īlways wear clean gloves or use forceps when handling membrane.Ĭover the membrane with liquid at all times to prevent drying. If the concentration of Tween 20 detergent is too high, it can strip proteins off the membrane. Increase the number of washes and/or the volume of buffer used.Īdd Tween 20 detergent to the wash buffer to a final concentration of 0.05%. Use Thermo Scientific SuperSignal Western Blot Enhancer to reduce background and enhance detection of low-abundance and weakly immunoreactive antigens. Prepare antibody dilutions in a blocking buffer that contains 0.05% Tween 20 detergent. For ease of use, choose a blocking buffer that already contains 0.05% Tween 20 detergent, such as Thermo Scientific StartingBlock T20 Blocking Buffer ( TBS) or ( PBS) or SuperBlock T20 Blocking Buffer ( TBS) or ( PBS). A final concentration of 0.05% often works well. However, too much detergent can interfere with antibody binding. Block for at least 1 hour at room temperature (RT) or overnight at 4☌.Īdding Tween 20 detergent to the blocking buffer can help minimize background. Optimize blocking time and/or temperature. Increase the concentration of protein in the blocking buffer. Insufficient blocking of nonspecific sites Use our blocking buffer selection guide to find the most compatible blocking buffer for your experiment. 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. Test for cross-reactivity in blocking buffer by blocking a clean piece of membrane, incubating with antibodies, and then detecting with the substrate of choice. Instead, block with BSA in Tris-buffered saline. When probing for phosphoproteins, avoid phosphate- based buffers like PBS and phosphoprotein-containing blockers like milk or casein. Milk contains biotin, which will result in high background. High concentration of RIPA (radioimmunoprecipitation assay) buffer results in widening of lanes and significant streaking during electrophoresisĭilute samples before electrophoresis to lower the final concentration of lysis buffer to prevent buffer-related defects.ĭecrease concentration of primary and/or secondary antibody.ĭo not use milk with avidin–biotin system. Use detergent removal columns or the Thermo Scientific Pierce SDS-PAGE Sample Prep Kit to remove excess detergent. Keep the ratio of SDS to nonionic detergent at 10:1 or greater to minimize these effects. Most of the nonionic detergents (e.g., Triton X-100, NP-40, and Tween 20 detergents) interfere with SDS- polyacrylamide gel electrophoresis (SDS-PAGE). High detergent concentration (e.g., SDS or Triton X-100 detergent) in gel electrophoresis.ĭetergents form mixed micelles with the anionic detergent SDS in the gel and migrate down into the gel they interfere with the SDS–protein binding equilibrium Make sure that the salt concentration does not exceed 100 mM. Use small-volume concentrators such as Pierce Protein Concentrators PES, 0.5 mL. Use a small dialysis device such as the Slide-A-Lyzer MINI Dialysis Device, 0.5 mL.Ĭoncentrate and resuspend samples in lower-salt buffer prior to electrophoresis. Perform dialysis to decrease salt concentration. High salt concentrations result in increased conductivity, which affects protein migration and can result in protein bands spreading into adjacent lanes containing samples with normal salt concentrations Excess salt (sodium chloride) in sample during gel electrophoresis.

Western protein ladder