Odyssey® Western Blot Detection:

Suggestions for Reducing Membrane Background and Nonspecific Binding

In some cases, it may be desirable to make minor modifications to the standard Odyssey Western blot procedure in order to reduce overall membrane background or nonspecific banding. These issues are most often encountered during detection in the 800 nm channel.

Changes in the detergents added to diluted antibody solutions, or in the choice of blocker itself, can greatly affect the quality of your results. If you are having difficulty with background in the 800 nm channel, wWe suggest that you make these following small modifications in your experimental procedure if you are having difficulty with background. Please note that the detergent recommendations for nitrocellulose and PVDF membranes are slightly different; follow the instructions for your membrane type. Modifications are compatible with both IRDyeTM800 and Alexa Fluor® 680 secondary antibodies.

Addition of SDS to Diluted Antibody Solutions

  • Adding a very small amount of SDS when you dilute your secondary antibody in blocker can help to eliminate nonspecific banding on your blots. In addition, it dramatically reduces the overall membrane background seen on PVDF membranes in the 800 nm channel. It is important to use a very small amount, because SDS can disrupt antibody-antigen interactions if too much is present at any time during the detection process. We recommend using a final concentration of 0.01 - 0.02% SDS. Some primary antibody/antigen complexes may be more sensitive to the presence of SDS in the secondary antibody incubation, so you may wishwant to try even lower concentrations in some cases.
  • If using PVDF membrane
  • No detergent should be present when blocking the blot, only during subsequent antibody incubations.
  • Primary antibody dilution: Add Tween-20 to a final concentration of 0.1% when the primary antibody is diluted in blocking buffer.
  • Secondary antibody dilution: Add both Tween-20 (to 0.1%) and SDS (to 0.01 - 0.02%) when diluting the secondary antibody in blocking buffer. SDS will greatly reduce overall membrane background on PVDF (which is often much darker than nitrocellulose membrane) and allow you to boost the image display settings to better view your bands. It can also reduce the appearance of nonspecific banding on the blot.
  • Proceed with washes as usual (using PBS + 0.1% Tween-20).
  • If using nitrocellulose membrane
  • No detergent should be present when blocking the blot, only during subsequent antibody incubations.
  • Primary antibody dilution: Add Tween-20 to a final concentration of 0.1 - 0.2% when the primary antibody is diluted in blocking buffer.
  • Secondary antibody dilution: Add both Tween-20 (to 0.1 - 0.2%) and SDS (to 0.01 - 0.02%) when diluting the secondary antibody in blocking buffer. SDS will help to reduce the appearance of nonspecific banding on the blot.
  • Proceed with washes as usual (using PBS + 0.1% Tween-20).
  • You can titrate the amount of SDS to find the level that provides maximum performance with your antibodies. These detergent recommendations have been tested with both Odyssey Blocker and casein blockers (see below).

Use of Casein Solutions for Blocking and Antibody Dilution

  • It is well known that no one blocking reagent will be optimal for every antibody-antigen pair. Some primary antibodies or antigens may display greatly reduced signal or different background bands in certain blockers, and there is no way to predict this behavior. If you are having difficulty detecting your target protein, it may be that changing the blocking buffer could greatly improve performance. This is often the case if you have had good luckresults with the antibody in the past using other blockers and detection systems in the past.
  • Odyssey Blocker and nonfat dry milk are two blockers commonly used for Odyssey detection. Casein has now been tested thoroughly as a blocking buffer and found to perform well. For some primary antibodies it may yield stronger signal, lower membrane background, and better specificity on the Odyssey system than other blockers. Casein should be used for both blocking and antibody dilutions.
  • Powdered casein from a number of sources has been tested, with little variation in performance. Hammersten-grade casein is not required. Casein blocker should be used at a concentration of 0.1% and should be made in 0.2 X PBS buffer. Higher concentration of PBS (up to 1 X) will also work, but 0.2 X appears to provide the highest signal. Do not make the buffer in water. A more concentrated stock solution may be made and diluted before use. To help dissolve the casein, you may autoclave the solution before use, if desired. EThe solution can be used even if all the particles do not dissolve the solution can be used. Pre-mixed 1% casein buffer concentrates in solution are available from Bio-Rad.
  • Follow the detergent recommendations above for use of SDS and Tween-20, for the appropriate membrane type.