SignalFire™ ECL Reagent is sensitive enough to detect picogram levels of protein. Western blot analysis of diluted recombinant human Akt-1 protein using Akt (pan) (C67E7) Rabbit mAb #4691.
|Petite Kit Includes||Quantity|
|SignalFire™ ECL Reagent A||1 x 25 ml|
|SignalFire™ ECL Reagent B||1 x 25 ml|
|Small Kit Includes||Quantity|
|SignalFire™ ECL Reagent A||1 x 250 ml|
|SignalFire™ ECL Reagent B||1 x 250 ml|
(a) Wash membrane-bound HRP (antibody conjugate) three times for 5 minutes in TBS/T.
(b) Prepare 1x SignalFireTM Plus ECL Reagent by diluting one part 2X Reagent A (#65293) and one part 2X Reagent B (#82201) (e.g. for 10ml, add 5ml Reagent A and 5ml 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).
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.
SignalFire™ ECL Reagent is stable at room temperature. DO NOT store Reagents A and B pre-mixed. Reagents A and B should be combined just prior to exposing membranes.
SignalFire™ ECL Reagent from Cell Signaling Technology (CST) is an enhanced chemiluminescent substrate capable of detecting picogram amounts of protein by western blot analysis. Compared to entry-substrates, 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.
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.