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View additional product information for SuperSignal™ West Pico PLUS Chemiluminescent Substrate - FAQs (34579, 34578, 34577, 34580X4, 34580, 34578X4)
17 product FAQs found
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).
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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).
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.
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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).
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.
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).
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.
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.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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).
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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).
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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).
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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).
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.