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View additional product information for SuperSignal™ ELISA Femto Substrate - FAQs (37074, 37075)
15 product FAQs found
A luminometer is used to measure relative light units.
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White plates provide the best signal-to-noise ratio. Black plates provide lower background but less detectable signal. Here are two suggestions we offer for white plates:
-Pierce 96-Well Polystyrene Plates, White Opaque (Cat. No. 15042)
-Nunc FluoroNunc/LumiNunc 96-Well Plates (Cat. No. 437796)
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Maximum signal is reached within two minutes and then declines steadily until it is neglible after twenty minutes.
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Our researchers have detected down to 168 fg of interleukin-2 in sandwich ELISA.
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We do not recommend diluting the working reagent. It is better to dilute or otherwise optimize primary and or secondary antibody concentrations or use more washes between incubation steps.
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The light emission wavelength is measured at 425 nm.
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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.
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We offer the Pierce Background Eliminator Kit (Cat. No. 21065) that reduces high background, shading, speckling, and over-exposed bands on exposed film.
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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.
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A high-quality membrane can effectively bind more target protein and result in a lower background after detection when using SuperSignal substrates.
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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.
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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).
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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.
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We have not tested the SuperSignal ELISA Femto Substrate with magnetic beads. However, we know that some metals can interfere with the substrate. Therefore, depending on the chemistry of the beads, they may or may not interfere with the substrate.
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Yes. The signal from the SuperSignal ELISA Femto Substrate ramps up very quickly and optimal signal production lasts only for 5-10 min. Therefore, we recommend using a multichannel pipette to add the substrate to the wells, mix for 1 min, and then read the signal produced. Typically, reading each well for 0.1-1 sec will provide good signal with this substrate.
Note: With the SuperSignal ELISA Pico Chemiluminescent Substrate (Cat. No. 37070, 37069), the signal does not decay as quickly and researchers typically have up to 30 min to read the plate.
Find additional tips, troubleshooting help, and resources within our Antibodies and Immunoassays Support Center.