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 Additional data should be considered for a more accurate adjustment.Excess salt (sodium chloride) in sample during gel electrophoresis. The molecular weight of each protein (kDa) was measured against an unstained protein ladder in every electrophoresis condition. Under suggested conditions, Unstained Protein Ladder, Broad Range resolves 12 major bands in 4-12% Bis- Tris Gel (MES buffer) and after Western blotting to the nitrocellulose membrane. 2.5 µl per well for general Western transferring.Ģ0 mM Tris-phosphate, pH 7.5 at 25☌, 2 % SDS, 0.2 mM Dithiothreitol, 3.6 M Urea, and 15 % (v/v) Glycerol.3 µl or 5 µl per loading for clear visualization during electrophoresis on 15-well or 10-well mini-gel, respectively.The ladder is supplied in the gel loading buffer and is ready to use. The Unstained Protein Ladder, Broad Range is designed for monitoring protein separation during SDS-polyacrylamide gel electrophoresis and verifying Western transfer efficiency on membranes (PVDF, nylon, or nitrocellulose) and for approximating the size of proteins. The proteins resolve into clearly identifiable bands when separated on the SDS-PAGE gel (MES buffer), with the intensified 25 kDa and 85 kDa protein bands serving as the two reference bands. The Unstained Protein Ladder, Broad Range is a mixture of 12 unstained recombinant proteins covering a wide range molecular weights from 10 to 200 kDa.

Western blot no protein or ladder