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View additional product information for SuperSignal™ West Dura Extended Duration Substrate - FAQs (34075X4, 34076X4, 34076, 34075, 37071)
21 product FAQs found
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).
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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).
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
See Tech Tip # 23: Strip and reprobe Western blots.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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).
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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).
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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).
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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).
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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).
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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).
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Yes. The SuperSignal Western blot substrates are well suited to most imaging systems including CCD camera units; however, most screen-type phosphorimagers give poor results with the SuperSignal substrates.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
We offer the Pierce Background Eliminator Kit (Cat. No. 21065) that reduces high background, shading, speckling, and over-exposed bands on exposed film.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Treating the nitrocellulose or PVDF membrane with Western Blot Signal Enhancer (Cat. No. 21050) before blocking can boost the chemiluminescent signal of low-intensity bands.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
A high-quality membrane can effectively bind more target protein and result in a lower background after detection when using SuperSignal substrates.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Nitrocellulose and PVDF membranes can be stripped following detection with SuperSignal chemiluminescent substrates, for reprobing. Restore Western Blot Stripping Buffer (Cat. No. 21059, 21063) can be used to gently but efficiently remove probes from the western blot membrane without damaging the target protein. In most cases, re-blocking of the membrane is not necessary.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
The most common cause of premature signal burnout is the use of excessive peroxidase-conjugated probe. During the substrate reaction, an extreme excess of peroxidase will deplete the limited amount of substrate resulting in intense, but short, signal duration. To avoid signal fading, it is imperative to use the peroxidase conjugated probe at a concentration recommended in the SuperSignal West Dura Extended Duration Substrate manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0011307_SupSig_West_Dura_Extend_Dur_Subs_UG.pdf).
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
The following are possible causes for weak signal intensity:
- Low level of protein transferred from the gel to the membrane. This is particularly true for high molecular weight proteins. Reversible Protein Stain Kits for Nitrocellulose and PVDF membranes (Cat. Nos. 24580 and 24585, respectively) may be used to stain the bands on the membrane to indicate transfer efficiency.
- Over-diluted primary and secondary antibody probes. We suggest using the recommended dilution for the antibody probes for each substrate. More sensitive substrates will require a more dilute antibody solution.
- Short substrate incubation time. For best results, incubate the membrane for 5-15 min in the SuperSignal substrate.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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.