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  5. Thermo Scientific™ SuperSignal™ West Pico PLUS Chemiluminescent Substrate

Thermo Scientific™ SuperSignal™ West Pico PLUS Chemiluminescent Substrate

Catalog No. PI34580
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Description

Thermo Scientific SuperSignal West Pico PLUS Chemiluminescent Substrate is an enhanced chemiluminescent (ECL) horseradish peroxidase (HRP) substrate that enables picogram to high femtogram-level protein detection by western blot analysis.

Thermo Scientific SuperSignal West Pico PLUS Chemiluminescent Substrate is an enhanced chemiluminescent (ECL) horseradish peroxidase (HRP) substrate that enables picogram to high femtogram-level protein detection by western blot analysis.

SuperSignal West Pico PLUS Chemiluminescent Substrate characteristics include:

  • Sensitivity: picogram to femtogram sensitivity
  • Stability: 8-hr working solution stability; 1-year kit stability at room temperature
  • Compatibility: nitrocellulose and PVDF membranes
  • Signal duration: 6 to 24 hours
  • Recommended primary antibody concentration: 1:1000–1:5000 dilution (0.2–1.0 μg/mL)
  • Recommended secondary antibody concentration: 1:20,000–100,000 dilution (10–50 ng/mL)

SuperSignal West Pico PLUS substrate is designed to provide excellent signal intensity and sensitivity for western blotting with HRP conjugates. The intensity of the light emission combined with the exceptional duration allows for the acquisition of multiple exposures to more easily obtain publication-quality blot images. SuperSignal West Pico PLUS substrate is compatible with different membranes, blocking reagents, and a wide range of antibody dilutions, making it an ideal choice for most western blotting applications.

Specifications

Product Type Substrate
Detection Method Chemiluminescence
Form Liquid
For Use With (Application) Western Blot
Membrane Compatibility Nitrocellulose, PVDF
Product Line SuperSignal
Quantity 500 mL
Conjugate HRP
Substrate Horseradish Peroxidase
Recommended Antibody Concentrations 2°: 1:20-100 K (10-50 ng/mL), 1°: 1:1 K (0.2-1 μg/mL)
Sensitivity Picogram to high femtogram-level
Signal Duration 6 to 24 hr.
Sufficient For 60 Mini-Gel Blots, 5000 cm2 of Membrane
Kit Contents Luminol/Enhancer, 250 mL, Stable Peroxide Buffer, 250 mL
Content And Storage Sufficient for 60 mini-gel blots; 5000 cm2 of membrane
• SuperSignal West Pico PLUS Luminol/Enhancer, 250 mL
• SuperSignal West Pico PLUS Stable Peroxide, 250 mL

Store at room temperature.
Shipping Condition Room Temperature
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Frequently Asked Questions (FAQs)

What types of membrane work best with SuperSignal West Pico PLUS Chemiluminescent Substrate?

Most researchers use nitrocellulose or polyvinyldiflouride (PVDF) membranes with SuperSignal West Dura Chemiluminescent 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).
Will SuperSignal West Pico PLUS Chemiluminescent Substrate work with nucleic acid blots (Southern blotting, etc.)?

Yes. However, SuperSignal West Pico PLUS Chemiluminescent 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).
I used the SuperSignal West Pico PLUS Chemiluminescent Substrate. Why is the entire film black or showing a reversed image?

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.
Why do I see more bands with the SuperSignal West Pico PLUS Chemiluminescent Substrate than I've seen in the past with other substrates?

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.
What protocol should I use to strip and reprobe a blot detected with SuperSignal West Pico PLUS Chemiluminescent Substrate ?

See Tech Tip # 23: Strip and reprobe Western blots.
Can the blots detected with the SuperSignal West Pico PLUS Chemiluminescent Substrate be reprobed?

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).
Should any reagents be avoided when using the SuperSignal West Pico PLUS Chemiluminescent Substrate?

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.
What is the best blocking reagent to use with SuperSignal West Pico PLUS Chemiluminescent Substrate?

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).
Is milk a good blocking reagent to use with SuperSignal West Pico PLUS Chemiluminescent Substrate?

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).
Can I use SuperSignal West Pico PLUS Chemiluminescent Substrate with the identical protocol (i.e., same dilutions) I used with the GE Healthcare ECL Substrate or Thermo Scientific ECL Western Blotting Subtrate (Cat. No. 32106)?

No, SuperSignal substrates require different dilutions and 5-minute incubation on the blot. You must use less (more dilute) primary and secondary antibodies with SuperSignal West Pico PLUS Chemiluminescent Substrate (see product instructions for recommended ranges). Please see Tech Tip # 67: Chemiluminescent Western Blotting Technical Guide and Protocols (https://tools.thermofisher.com/content/sfs/brochures/TR0067-Chemi-Western-guide.pdf).
How can I increase the sensitivity of my Western blot?

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.
How do I make the working solution for the SuperSignal West Pico PLUS Chemiluminescent Substrate?

To prepare the working solution for the SuperSignal West Pico PLUS Chemiluminescent Substrate, follow these steps:

- Mix equal parts of the SuperSignal West Pico PLUS Luminol/Enhancer Solution and SuperSignal West Pico PLUS Stable Peroxide Solution. For example, for a mini blot, mix 5 mL of SuperSignal West Pico PLUS Luminol/Enhancer Solution with 5 mL of SuperSignal West Pico PLUS Stable Peroxide Solution.
- Ensure that the volume is sufficient to completely wet the blot and prevent it from drying out. The recommended volume is 0.1 mL/cm2.
- Note that the working solution is stable for up to 8 hours at room temperature. Avoid exposure to the sun or other intense light. Short-term exposure to lab lighting is okay.
How does SuperSignal West Pico PLUS Chemiluminescent Substrate compare with competitors' products?

SuperSignal West Pico PLUS Chemiluminescent Substrate has a better sensitvity than Bio-Rad's Clarity. Also, compared to Millipore's Immobilon substrate, SuperSignal West Pico PLUS Chemiluminescent Substrate has a better signal duration over 4 hours. Please also see Tech Tip # 32 Guide to Enzyme Substrates for Western Blotting (https://tools.thermofisher.com/content/sfs/brochures/TR0032-Substrates-Western.pdf).
When should I use SuperSignal West Pico PLUS Chemiluminescent Substrate?

SuperSignal West Pico PLUS Chemiluminescent Substrate can be used as an everyday substrate. For assays requiring higher sensitivity, we recommend using SuperSignal West Dura Extended Duration Substrate or SuperSignal West Femto Maximum Sensitivity Substrate.
What is the sensitivity of SuperSignal West Pico PLUS Chemiluminescent Substrate?

SuperSignal West Pico PLUS Chemiluminescent Substrate enables detection of low-picogram to high-femtogram amounts of target protein on nitrocellulose or PVDF membrane. Please also see Tech Tip # 32 Guide to Enzyme Substrates for Western Blotting (https://tools.thermofisher.com/content/sfs/brochures/TR0032-Substrates-Western.pdf).
What is the recommended dilution range for SuperSignal West Pico PLUS Chemiluminescent Substrate?

Primary antibody recommended dilution range is 0.2 to 1.0 µg/mL (1:1000 to 1:5000 dilution from a 1 mg/mL stock). Secondary antibody recommended dilution range is 10 to 50 ng/mL (1:20,000 to 1:100,000 dilution from 1 mg/mL stock). Please also see Tech Tip # 32 Guide to Enzyme Substrates for Western Blotting (https://tools.thermofisher.com/content/sfs/brochures/TR0032-Substrates-Western.pdf).
How does SuperSignal West Pico PLUS Chemiluminescent Substrate compare with the original SuperSignal West Pico Chemiluminescent Substrate?

SuperSignal West Pico PLUS Chemiluminescent Substrate has a higher sensitivity and greater forgiveness for broad dilution range compared to SuperSignal West Pico Chemiluminescent Substrate. Please also see Tech Tip # 32 Guide to Enzyme Substrates for Western Blotting (https://tools.thermofisher.com/content/sfs/brochures/TR0032-Substrates-Western.pdf).

For Research Use Only. Not for use in diagnostic procedures.