Product Name :BiodlightTM Biodlight Western Chemiluminescent HRP Substrate
Applications :Western Blot
Background :BiodlightTM ECL Chemiluminescent HRP Substrate provides high sensitivity in western or dot/slot/spot blotting applications on both PVDF and nitrocellulose transfer membranes,and is compatible with all commonly used buffers and blocking reagents.The HRP substrate consists of Luminol Reagent and Perixide Solution.Working HRP substrate is prepared by combining equal volumes of Luminol Reagent and Perixide Solution.The HRP substrate produces a high intensity signal with low background for detection of both high and low abundance.
Classification :WB Related Reagents
Contents :Luminol Reagent,50ml Peroxide Solution,50ml
Storage & Shelf Life :Store at 4° C
Procedure :1. Remove blot from the transfer apparatus and block nonspecific sites with Blocking Reagent for 20-60 minutes at room temperature (RT) with shaking. For best results, block for 1 hour at RT.2. Remove the Blocking Reagent and add the appropriate primary antibody dilution. Incubate blot for 1 hour with shaking.If desired, blots may be incubated with primary antibody overnight at 2-8°C.3. Wash membrane by suspending it in Wash Buffer and agitating for 5 minutes. Replace Wash Buffer at least 4-6 times. Increasing the wash buffer volume and/or the number of washes may help reduce background.Briefly rinsing membrane in wash buffer before incubation will increase wash efficiency.4.Incubate blot with the appropriate HRP-conjugate dilution for 1 hour at RT with shaking.5.Repeat Step 3 to remove non-bound HRP-conjugate.Membrane must be thoroughly washed after incubation with the HRP-conjugate.6.Prepare Working Solution by mixing equal parts of the Stable Peroxide Solution and the Luminol/Enhancer Solution. Use 0.1 ml Working Solution per cm2 of membrane. The Working Solution is stable for 8 hours at room temperature.Exposure to the sun or any other intense light can harm the Working Solution. For best results keep the WorkingSolution in an amber bottle and avoid prolonged exposure to any intense light. Typical laboratory lighting will not harm the Working Solution.7.Incubate blot with Working Solution for 5 minutes.8.Remove blot from Working Solution and place it in a plastic membrane protector; a plastic sheet protector or plastic wrap may be used. Use an absorbent tissue to remove excess liquid and to carefully press out any bubbles from between the blot and surface of the membrane protector.9.Place the protected membrane in a film cassette with the protein side facing up. Turn off all lights except those appropriate for film exposure (e.g., a red safelight).Film must remain dry during exposure. For optimal results, perform the following precautions:• Make sure excess substrate is removed from the membrane and the membrane protector.• Use gloves during the entire film-handling process.• Never place a blot on developed film, as there may be chemicals on the film that will reduce signal.10.Carefully place a piece of film on top of the membrane. A recommended first exposure time is 60 seconds. Exposure time may be varied to achieve optimal results. Enhanced or pre-flashed film is not necessary.Light emission is intense and any movement between the film and membrane can cause artifacts on the film.The exposure time may be varied to achieve optimal results. If the signal is too intense, reduce exposure time or optimize the system by decreasing the antigen and/or antibody concentrations.Light emission is most intense during the first 5-30 minutes after substrate incubation. Light emission will continue for several hours, but will decrease with time. Longer exposure times may be necessary as the blot ages.11.Develop film using appropriate developing solution and fixative. Blot may be stripped and reprobed if necessary.
Research use :For research use only, not for use in diagnostic procedures.
Expiration Date :One year from date of reconstitution.
