Description
Product Usage 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. 1. Wash membrane-bound HRP (antibody conjugate) three times for 5 minutes in TBS/T: (20 mM Tris-HCl (pH 7.6),137 mM NaCl and 0.1% Tween-20). It is very important to thoroughly wash the membrane prior to substrate incubation. 2. Prepare 1x SignalFire™ Plus ECL Reagent by diluting one part 2X Reagent A (#65293) and one part 2X Reagent B (#82201) (e.g., for 10 ml, add 5 ml Reagent A and 5 ml Reagent B). Mix well. 3. 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).
Storage
Store at RT. This product is stable for 36 months. DO NOT store Reagents A and B pre-mixed. Reagents A and B should be combined just prior to exposing membranes.
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.
Alternate Names
Chemiluminescence; ECL; HRP; Luminol; Signal Fire; Substrate
