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 Simply trace the visible markers on the membrane after transfer and before blocking (Figure 2).įigure 2.Excess salt (sodium chloride) in sample during gel electrophoresis. Marking the location of visible protein standards on a blot is easy. The WesternBright ChemiPen has two tips, a fine tip at one end that is perfect for writing on the blot or tracing the bands of a visible protein standard, and a thicker wedge tip that can be used to deposit greater amounts of reagent on the blot. The WesternBright ChemiPen can be used to mark the location of protein standards and to annotate the blot with a date, as in the right panel. With the proprietary "ink" you can transform your visible protein markers into chemiluminescent markers, to annotate your blot with a date or blot ID, or to check the stability of your HRP substrate (Figure 1).įigure 1. The reagent in the ChemiPen adsorbs to nitrocellulose and PVDF membranes, and reacts with HRP substrates to produce chemiluminescence that can be detected with X-ray film or CCD imaging. Write or draw on your transfer membranes with the WesternBright ® ChemiPen.
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