Detection Systems
SignalFire™ ECL Reagent #6883
Description
SignalFire™ ECL Reagent from Cell Signaling Technology (CST) is an enhanced luminol based system designed for use with CST™ HRP-linked secondary antibodies and Phototope®-HRP detection assays. SignalFire™ ECL Reagent is extremely sensitive and capable of detecting protein levels at low picogram amounts. Compared to other luminol based systems, SignalFire™ ECL Reagent boasts a more robust signal and extended duration of signal output. In the presence of hydrogen peroxide, horseradish peroxidase (HRP) converts luminol to an excited intermediate dianion. This dianion emits light on return to its ground state. Light emission is maximal immediately after exposure of the substrate to HRP and continues for 1 hour. Light can be captured on X-ray film, typically by exposure for a few seconds. Maximum sensitivity can be obtained with longer exposure.
Western Blotting
SignalFire™ ECL Reagent is sensitive enough to detect low picogram levels of protein. Western blot analysis of diluted recombinant human HO-1 protein (hHO-1) using HO-1 (D60G11) Rabbit mAb #5853.
Directions for Use
(a) Wash membrane-bound HRP (antibody conjugate) three times for 5 minutes in TBS/T.
(b) Prepare 1X SignalFire™ ECL Reagent by diluting one part 2X Reagent A and one part 2X Reagent B (e.g. for 10 ml, add 5 ml Reagent A and 5 ml Reagent B). Mix well.
(c) Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.
*Avoid repeated exposure to skin (see enclosed Material Safety Data Sheet or refer to our website for further information).
Solutions and Reagents
Each SignalFire™ ECL Reagent (A and B) is a 2X concentrate; there is no need to further dilute in water when the two reagents are combined.
Background
Chemiluminescence systems have emerged as the best all-around method for western blot detection. They eliminate the hazards associated with radioactive materials and toxic chromogenic substrates. The speed and sensitivity of these methods are unequalled by traditional alternatives, and because results are generated on film, it is possible to record and store data permanently. Blots detected with chemiluminescent methods are easily stripped for subsequent reprobing with additional antibodies. HRP-conjugated secondary antibodies are utilized in conjunction with specific chemiluminescent substrates to generate the light signal. HRP conjugates have a very high turnover rate, yielding good sensitivity with short reaction times.
Application References
Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!
Companion Products
- 7071 Phototope®-HRP Western Blot Detection System, Anti-rabbit IgG, HRP-linked Antibody
- 7072 Phototope®-HRP Western Blot Detection System, Anti-mouse IgG, HRP-linked Antibody
- 7074 Anti-rabbit IgG, HRP-linked Antibody
- 7075 Anti-biotin, HRP-linked Antibody
- 7076 Anti-mouse IgG, HRP-linked Antibody
- 7077 Anti-rat IgG, HRP-linked Antibody
For Research Use Only. Not For Use In Diagnostic Procedures.
