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Efficient chemiluminescence kitCat # Product Name Component Pack SizeGE2301-25MLEfficient chemiluminescence kitWestern HRP SubstrateLuminol Reagent12.5ml×2250cm2 GE2301-50ML 25ml×2500cm2 Western HRP SubstratePeroxide Solution GE2301-100ML 50ml×21000cm2 IntroductionChemiluminescent detection uses an enzyme to catalyze a reaction that results in the generation of visiblelight. The horseradish peroxidase(HRP) chemluminescent reaction is based on the catalyzed oxidation of luminolby peroxide.Oxidized luminol emits light as it decays to its ground state. This technique has the speed and safetyof chromogenic detection methods, at higher sensitivity levels.Western HRP Substrate provides high sensivity in western or dot/slot/spot blotting application on bothPVDF and nitrocellulose transfer membranes, and is compatible with all commonly used buffers and blockingreagents. Blots on PVDF membrane may be reprobed, allowing detection of multiple target proteins on the same blot.The HRP substrate consists of Luminol Reagent and Peroxide Solution. Working HRP substrate isprepared by combining equal volumes of Luminol Reagent and Peroxide Solution. The HRP substrate produces a high intensity signal with low background for detection of both high and low background for detection of bothhigh and low abundance proteins.Materials Required for Western Blotting1. PVDF membrane or nitrocellulose membrane.2. Sheets of filter paper, cut to dimension of the gel.3. 100% methanol (to pre-wet PVDF membrane).4. Transfer system and transfer buffer.5. ddH2O water.6. Wash buffer: Phosphate-buffered saline(PBS) or Tris-buffered saline(TBS) containing 0.05-0.1% Tween-20surfactant; (PBS:10 mM sodium phosphate, 150 mM NaCl, pH7.2. TBS: 10 mM Tris, 150 mM NaCl,pH7.4).7. Blocking buffer:1-5%(w/v) blocking agent (e.g., casein, BSA, or nonfat dry milk ) in wash buffer.NOTE: Western HRP Substrate is compatible with all blocking buffer.2% casein solution isrecommended for the lowest background and highest signal-to-noise ratio.8. Primary antibody specific for the protein of interest, diluted in wash buffer or blocking buffer.9. HRP-conjugated secondary antibody, specific for primary antibody, diluted in wash buffer or blockingbuffer.10. Shallow trays, large enough to hold the blot.11. Plastic wrap, plastic bag, transparency or sheet protector.12. X-ray film and developer reagents or chemiluminescence-compatible imaging systems.Usage Guidelines1. Due to the high sensitivity of the Western HRP Substrate, lower amounts of antigen and higher dilutions ofprimary and secondary antibodies are recommended.Typical primary antibody dilutions are 1:1000-1:20000and secondary antibody dilutions typically range from 1:20000-1:200000. Important: If switching toWestern HRP Substrate from a lower sensitivity substrate, previous antibody dilution factors mayneed to be increased at least five-fold for the primary antibody and two-to five-fold for the secondaryantibody to achieve the optimal signal-to-noise ratio.2. Optimization of blocking reagents and incubation times will improve results and should be determinedexperimentally.3. The high sensitivity of the Western HRP Substrate may result in a significant reduction in required x-ray filmexposure time. An initial exposure time of 30 seconds is recommended. Optimum exposure time should bedetermined for each antibody system.4. Always wear gloves and use blunt tip forceps when handling the membrane to avoid contamination.5. Use care when handling the membrane to prevent tearing.6. Do not use sodium azide, which inhibits HRP activity, in any buffer or reagents.7. Use of blocking buffer to dilute antibodies may reduce background and increase sensitivity.Western Blotting ProtocolProtein Transfer1. Resolve the protein mixture on a 1-D or 2-D polyacrylamide gel.2. Immerse the gel in an appropriate transfer buffer and allow it to equilibrate for 10-15 minutes.3. If working with a PVDF membrane: Wet the membrane in 100% methanol for 15 seconds, or until themembrane appearance changes uniformly from opaque to semitransparent.If working with a nitrocellulose membrane: Proceed to step 4. Nitrocellulose do not require prewetting.4. Equilibrate the membrane for at least 5 minutes in the transfer buffer.5. Soak filter paper in the transfer buffer for at least 30 seconds.6. Assemble the transfer stack as shown below.NOTE: To ensure an even transfer, remove air bubbles by carefully rolling a clean pipette over the surfaceof each layer in the stack. Avoid excessive pressure that can damage the gel and membrane.----Filter paper Three Layers----Gel----Membrane----Filter paper Three Layers7. Transfer proteins according to blotting apparatus manufacturer’s instructions.8. Remove the blot from the transfer system and briefly rinse the membrane in ddH2O water. to remove geldebris. Proceed with immunodetection protocol below. If required, the PVDF membrane blot may be airdried and stored refrigerated for several months.Antibody Incubations1. If PVDF membrane were dried after transfer, wet the blots in 100% methanol for 15 seconds, or untilthe membrane appearance changes uniformly from opaque to semitransparent.NOTE: Omit this step if using nitrocellulose membrane.2. Rinse the blot with water and then place the blot in blocking buffer and incubate for 1 hour with gentleagitation at room temperature.3. Prepare primary antibody solution by diluting the antibody in wash or blocking buffer. See UsageGuidelines for antibody dilutions.4. Place the blot in the diluted primary antibody solution and incubate for at least 1 hour with gentleagitation. Ensure that the solution moves freely across the entire surface of the membrane.5. Wash the blot with fresh wash buffer a minimum of three times with gentle agitation for 5-10 minutes.Additional or longer washes may further reduce background. Immobilon-p (0.2μm) membrane mayrequire additional washing due to its greater surface area. 6. Prepare HRP-conjugated secondary antibody solution by diluting the antibody in wash or blockingbuffer. See Usage Guidelines for antibody dilutions.7. Place the blot in the diluted HRP-conjugated secondary antibody solution, and incubate for 1 hour withgentle agitation. Ensure that the solution moves freely across the entire surface of the membrane.8. Wash the blot with fresh wash buffer a minimum of three times with gentle agitation for 5-10 minutes.Additional or longer washes may further reduce background.Chemiluminscent detection1. To prepare working HRP substrate, mix equal volumes of Luminol Reagent and Peroxide Solution in aclean container or test tube. Approximately 0.1 mL of working HRP substrate is required per cm2 membrane area. The volumes of working HRP substrate needed for some common membrane sizes are indicated below:Blot Size Working HRP Substrate Required7×8.5cm 6ml (3ml Luminol reagent)(3ml peroxide solution)10×10cm 10ml (5ml Luminol reagent)(5ml peroxide solution)8.5×13.5cm 12ml (6ml Luminol reagent)(6ml peroxide solution)2. Allow the HRP substrate to reach room temperature (～10 minutes). Protection from light is notrequired.3. Place the blot protein side up in a clean container, and add the HRP substrate onto the blot.4. Incubate the blot for 5 minutes at room temperature.5. Drain the excess substrate.6. Cover the blot with a clean plastic wrap or sheet protector and remove any air bubbles. Ensure that thesurface of the plastic wrap or sheet protector is dry and unwrinkled.7. Expose the blot to a suitable X-ray film for an appropriate duration. Because of the high sensitivity ofthe Western HRP Substrate, a shorter exposure time may be required. The recommended initialexposure time is 30 seconds. The chemiluminescent,signal on the blot will last at least two hours. Ifnecessary, fresh HRP substrate can be added to the same blot for consecutive exposures.NOTE: The working HRP substrate can be stored up to 7 days in the dark at 2-8℃ without anydetectable loss of activity.Membrance Stripping （Only for PVDF Membrances） A single blot on PVDF membrane can be sequentially probed by stripping the first antibody from the blot,and then incubating with a different primary antibody. This is especially useful for method optimization or whensample amount is limited.The stripping process disrupts the antigen-antibody interaction,usually by a combination of detergent and heat or by exposure to low pH.