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.
Kit Components:Reagent A, 2x 1x250mlReagnet B, 2x 1x250ml
Protocol:1. Wash membrane-bound HRP (antibody conjugate) three times for 5 minutes in TBS/T.
2. Prepare 1X ECL Reagent by diluting one part 2C3900-03A: Reagent A, 2x and one part C3900-03B: Reagnet B, 2x (e.g. for 10 ml, add 5ml Reagent A and 5ml 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.
Storage and Stability:May be stored at RT. Stable as 2x for 6 months.
