SuperSignal™ West Dura Extended Duration Substrate, 200 mL - FAQs

Most researchers use nitrocellulose or polyvinyldiflouride (PVDF) membranes with SuperSignal West Dura Extended Duration Substrate. Both work well, although nitrocellulose seems to be better suited in some applications than PVDF. In addition, charge-modified nylon membrane performs well with this substrate. Please also see our guide for choosing western blot membranes (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/western-blotting/transfer-proteins-western-blot/membranes-transfer-buffers-western-blotting/membranes-western-blotting.html).

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Yes. However, SuperSignal West Dura Extended Duration Substrate was optimized for use in Western blots and is generally not sensitive enough for most nucleic acid protocols. For Southern and Northern blotting, use our North2South Chemiluminescent Nucleic Acid Hybridization and Detection Kit (Cat. No. 17097), North2South Chemiluminescent Substrate for HRP (Cat. No. 17295), or the Chemiluminescent Nucleic Acid Detection Kit, if probing for biotinylated probes (Cat. No. 89880).

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The antibody concentration (primary and secondary) is too high. Use the antibody dilutions described in the product instructions. Most background will disappear when the proper blocking reagent and a HRP conjugate concentration are used.

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Because this substrate is more sensitive than other chemiluminescent substrates you might detect low-abundance proteins that were not visible before. When using a more sensitive substrate, more careful optimization of blocking buffer steps and antibody concentrations is required.

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See Tech Tip # 23: Strip and reprobe Western blots.

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Although blots detected with chemiluminescent substrates can be stripped and reprobed, some antigen/antibody systems are sensitive to the stripping procedure and might not yield the same quality of results on a stripped blot compared to a new blot. Only actual experimentation will yield information as to whether a given system will allow reprobing. Please also see Tech Tip # 23 Strip and Reprobe Western Blots (https://tools.thermofisher.com/content/sfs/brochures/TR0023-strip-reprobe-blots.pdf).

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The lower detection limit of SuperSignal West Dura Extended Duration Substrate is mid-femtogram (1 x 10^-15). Please also see Tech Tip # 32 Guide to Enzyme Substrates for Western Blotting (https://tools.thermofisher.com/content/sfs/brochures/TR0032-Substrates-Western.pdf).

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Yes. Avoid using sodium azide during and after probing steps involving horseradish peroxidase (HRP), as this will inhibit HRP activity. Sulfide, cyanide, fluoride and superoxide ions also inhibit HRP to some extent.

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Background is a relative phenomenon - no blocker will prevent all interactions 100% of the time. While a particular blocking agent may give a perfect signal-to-noise ratio for one set of reaction conditions, it may not work as well for another set of similar conditions. The key is to optimize the system by trying various blocking conditions. Please see Tech Tip # 22 Determine source of non-specific background signal in Western Blots (https://tools.thermofisher.com/content/sfs/brochures/TR0022-Determine-background-cause.pdf).

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Yes and no. Milk contains variable amounts of biotin so it should not be used with avidin/biotin detection systems. Milk also contains varying amounts of phosphoproteins that may make interfere with anti-phosphotyrosine procedures. A variety of blocking buffers are compatible with the substrate. Please see our Blocking Buffer selection table (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/western-blotting/detect-proteins-western-blot/western-blot-buffers/blocking-buffers-western-blotting.html#table).

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SuperSignal West Dura Extended Duration Substrate is about 10-fold more sensitive than GE Healthcare ECL Substrate or Thermo Scientific ECL Substrate (Cat. No. 32106). SuperSignal West Dura Extended Duration Substrate is also significantly more sensitive than available chemiluminescent substrates for alkaline phosphatase. Please also see Tech Tip: Guide to Enzyme Substrates for Western Blotting (https://assets.thermofisher.com/TFS-Assets/LSG/Application-Notes/TR0032-Substrates-Western.pdf).

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Consider transferring to a different membrane or using a different detection method. We have observed increased sensitivity when using PVDF membranes in place of nitrocellulose. On PVDF membranes, using as little as 1 µg of total rat brain protein, PKC can be detected with alkaline phosphatase-mediated chromogenic detection in some cases using affinity-purified antibodies at a concentration of 0.5 µg/mL. Detection sensitivity can also be increased by using chemiluminescent detection, especially when using a SuperSignal West enhanced chemiluminescence subtrate (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/western-blotting/detect-proteins-western-blot/western-blot-detection-reagents/detection-technologies-western-blotting/chemiluminescent-western-blot-detection/supersignal-chemiluminescent-substrates.html) such as SuperSignal West Pico PLUS, SuperSignal West Dura, or SuperSignal West Femto. The secondary antibody should be used as recommend by the manufacturer and optimized as needed.

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No, these SuperSignal substrates require different dilutions. If you follow the SuperSignal West Pico Chemiluminescent Substrate protocol when you use SuperSignal West Dura Extended Duration Substrate, you will see high background or a decreased signal intensity and or duration. You must use less (more dilute) primary and secondary antibodies with SuperSignal West Dura Extended Duration Substrate (see product instructions for recommended ranges).

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In the darkroom, prepare 2 mL of working substrate solution by mixing 1 mL of luminol enhancer and 1 mL of stable peroxide in a clear test-tube. Add 1 µL of undiluted HRP-conjugate to the working substrate solution. The solution should immediately emit a blue light that will fade over the next several minutes. This glow indicates that the components in the substrate kit are still active/functional. If no light emission is evident, test another source of HRP to determine the root cause.

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The following are possible causes for high background signal:
- Insufficient blocking of the membrane
- Using a non-optimized blocker
- Using excess peroxidase-conjugated probe
- Cross-reactivity of the probes to the blocking agent

We recommend testing a variety of blockers to find the best one for a given system.
- Serum-based blockers are a popular choice but have the potential to cross-react with an antibody probe.
- Avoid blocking the membrane with BSA when using an antibody against a bovine source.
- Follow the recommended dilution of the peroxidase-conjugated probe for the chemiluminescent substrate selected to reduce the potential for excessive background.

The following tech tips provide more information that may be useful:
- https://assets.thermofisher.com/TFS-Assets/LSG/Application-Notes/TR0022-Determine-background-cause.pdf
- https://assets.thermofisher.com/TFS-Assets/LSG/Application-Notes/TR0024-Optimize-Ab-dilutions.pdf
- https://assets.thermofisher.com/TFS-Assets/LSG/Application-Notes/TR0032-Substrates-Western.pdf
- https://assets.thermofisher.com/TFS-Assets/LSG/Application-Notes/TR0067-Chemi-Western-guide.pdf

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

SuperSignal West Dura Extended Duration Substrate is a luminol-based enhanced chemiluminescence (ECL) horseradish peroxidase (HRP) substrate. It is oxidized to an excited state product in the presence of the stable peroxide and horseradish peroxidase. As the product decays to a lower energy state, light is released at 425 nm. The emitted light is then captured on X-ray film or by using a CCD camera.

SuperSignal West Dura Extended Duration Substrate for HRP is optimized for high sensitivity and long signal duration, making it ideal for cooled CCD camera-based detection systems. Unlike substrates with signals that decline to barely detectable levels in 30-60 min, the signal produced with SuperSignal West Dura Extended Duration Substrate is stable for 24 hr, allowing multiple film or camera exposures.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.