Knowing when and what concentration of detergents to use is critical to Western blotting success. Two detergents, sodium dodecyl sulfate (SDS) and Tween® 20, can help to reduce background signals and promote specific antibody binding to the target. However, overdosing your blot with detergents or using the wrong one can result in lowered signal-to-noise ratios. The type of membrane also influences the concentration of detergent that should be used. Make sure you know when detergents are helpful and when they should be left out for best results.
Blocking Buffer
Blocking buffers can reduce background signals and promote specific antibody binding to the target; however, too much of it can lower signal-to-noise ratios. For both chemiluminescent and fluorescent blots, detergents should not be added during the blocking step, and blots should not be exposed to detergents until blocking is complete. Otherwise, it can cause higher background autofluorescence by decreasing blocking efficiency.
Primary Antibody Dilution
Both nitrocellulose and low-fluorescence PVDF membranes should use a final concentration of 0.1-0.2% Tween 20. Keep in mind that for near-infrared (NIR) fluorescence, low-fluorescence PVDF membranes are essential for optimal results. Do not use SDS with either membrane during primary antibody dilution. Intercept® T20 Antibody Diluents can be used in primary antibody dilutions and improve the primary antibody’s binding affinity to the antigen.
Secondary Antibody Dilution
In a secondary antibody dilution, use a final concentration of 0.1-0.2% Tween 20 for both nitrocellulose and low-fluorescence PVDF membranes. For nitrocellulose, do not use SDS; for PVDF, add 0.01-0.02% SDS to the final antibody dilution. Intercept T20 Antibody Diluents can also be used to improve the specificity of secondary antibodies and improve their binding affinity to the antigen.
Wash Solution
For nitrocellulose and low-fluorescence PVDF membranes, add 0.1% Tween 20 to the wash solution. Do not add SDS for either membrane.
Detergents and Western Blotting Summary
Step | Nitrocellulose membranes | Low-fluorescence PVDF membranes |
---|---|---|
Blocking buffer | Do not add detergents to blocking buffer. | Do not add detergents to blocking buffer. |
Primary antibody dilution | Use a final concentration of 0.1 - 0.2% Tween® 20. Do not use SDS with nitrocellulose membranes. | Use a final concentration of 0.1 - 0.2% Tween® 20. Do not use SDS with nitrocellulose membranes. |
Secondary antibody dilution | Use a final concentration of 0.1 - 0.2% Tween® 20. Do not use SDS with nitrocellulose membranes. | Use a final concentration of 0.1 - 0.2% Tween® 20. Add 0.01 - 0.02% SDS to final antibody dilution. |
Wash solution | Add 0.1% Tween® 20. Do not use SDS with nitrocellulose membranes. | Add 0.1% Tween® 20. Do not add SDS to wash solution. |
We encourage you to visit our Reagent Store to explore our wide variety of reagents and learn more about detergents used in Western blotting. If you have questions or concerns about detergents, Western blotting, or reagents in general, then please don't hesitate to contact us today. We'd love to discuss how our reagents and imaging systems can help you optimize your lab's Western blotting protocols.