SuperSignal™ West Dura Extended Duration Substrate, 100 mL - FAQs

大多数研究人员使用硝化纤维素或PVDF膜以用于SuperSignal West Dura化学发光底物。两种效果都很好，但是硝化纤维素膜在一些应用中比PVDF膜更合适。此外，对于这种底物，荷电尼龙膜效果较好。

< p >是的。然而，SuperSignal West Dura化学发光的底物是为Western blots优化的，通常对大多数核酸操作不够敏感。对于Southern和Northern blotting, 使用我们的North2South化学发光核酸杂交和检测试剂盒(货号17097)，用于HRP的North2South化学发光底物（货号17097）或者如果用生物素化探针杂交的话，使用化学发光核酸检测试剂盒(货号 89880)。< / p >

抗体浓度（一抗和二抗）过高。按照产品说明稀释抗体。当使用了合适的封闭剂和HRP结合物浓度时，大部分背景会消失。

因为这种底物比其他化学发给底物更灵敏，使您能够检测到之前看不到的低丰度蛋白。当使用更灵敏的底物时，需要对封闭液和抗体浓度进行更仔细的优化。

请查看“技术贴士#23：剥离和再次检测免疫印迹膜”(https://tools.thermofisher.com/content/sfs/brochures/TR0023-strip-reprobe-blots.pdf)中的实验方案。

尽管使用化学发光底物检测过的印迹膜可剥离并再次检测，但是，有些抗原/抗体系统对剥离步骤较为敏感，剥离后的印迹膜可能不会产生与新印迹膜质量相同的结果。只有经过实际实验，才能知道某个系统是否可进行再次检测。

SuperSignal West Dura化学发光底物的检测下限为中飞克级（1 x 10^–15）。

有。在涉及辣根过氧化物酶（HRP）的检测步骤期间和之后，应避免使用叠氮化钠，因为它会抑制HRP活性。硫化物、氰化物、氟化物和超氧离子也会在一定程度上抑制HRP。

背景是一种相对的现象——没有一种封闭剂总是能够防止所有相互作用的发生。尽管一种特殊的封闭剂可能会在一组反应条件下产生完美的信噪比，但可能对另一组相似条件无效。关键在于通过改变封闭条件对系统进行优化。

是，也不是。牛奶的生物素含量是可变的，因此不可用于抗生物素蛋白/生物素检测系统。牛奶的磷蛋白含量也是可变的，因此可能干扰抗磷酸酪氨酸过程。有多种封闭剂溶液可兼容SuperSignal化学发光底物。

SuperSignal West Dura化学发光底物的灵敏度比GE Healthcare ECL底物或Thermo Scientific ECL底物（货号32106）高10倍。SuperSignal West Dura化学发光底物的灵敏度也显著高于碱性磷酸酶的化学发光底物。

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.

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.

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.

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.

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.