Hypersensitive ECL Chemiluminescent Substrate Reagent
Hypersensitive ECL Chemiluminescent Substrate
Synonyms
Hypersensitive ECL Chemiluminescent Substrate; Hypersensitive ECL Chemiluminescent Substrate (RTU); Hypersensitive ECL Chemiluminescent Substrate reagent
Protocol
1. Proceed the routine SDS-PAGE, transfer membrane and Western Blot steps.
2. Blot membrane: wash membrane with TBS-T twice, 10 min each. Immerse membrane into 5% defatted milk blocking solution, shake by a shaker, and incubate at room temperature for 1 hour.
3. Remove the blocking solution, incubate the membrane in properly diluted primary antibody at room temperature for 2 hours, or at 4? overnight on a shaker.
Note: Dilute the primary antibody with blocking solution at the antibody manufacturer's recommended dilution.
4. Wash the membrane with 4× or 6× TBS-T for 3 times, 10 min each. Increase the volume of wash buffer, and / or the wash times can reduce the background.
Note: Before proceeding Step 4, rinse the membrane with TBS-T simply, the wash efficiency will be increased.
5. Incubate the membrane in properly diluted HRP conjugated secondary antibody at room temperature for 2 hours on a shaker.
Note: Dilute the secondary antibody at the antibody manufacturer's recommended dilution.
6. Repeat Step 4 to remove the non-specific conjugation of HRP conjugated secondary antibody.
Note: Wash the membrane thoroughly after proceeding Step 5.
7. Prepare substrate working solution: mix Reagent A with Reagent B at ratio 1:1, i.e. Mix 1 ml of Reagent A with1 ml of Reagent B, mix thoroughly for use. The dosage of this working solution should be determined according to the membrane size, it is better to immerse the membrane completely in this working solution, i.e. Per 10 cm2 membrane need about 1 ml of this working solution.
8. Put the membrane on a flat (protein side upwards), add the substrate working solution.
9. Incubate the membrane with substrate working solution for 1-5 min.
Note: Observe the incubation in the dark room to determine whether need to do film exposure.
10. Spread a plastic wrap or transparent glassine on the membrane, if the plastic wrap or transparent glassine is big enough, it is unnecessary to remove the excess working solution, but just need slightly press out the bubbles.
11. In the dark room, carefully put a piece of X-ray film on top of the membrane, expose 5 seconds to 1 minute, develop and fix film immediately.
Note: The exposure time can be varied according to chemiluminescence idensity to achieve optimal results. If the signal is weak, prolong the exposure time to hours. Or use a gel visualization or a CCD camera to record chemiluminescent images of the membrane directly.
2. Blot membrane: wash membrane with TBS-T twice, 10 min each. Immerse membrane into 5% defatted milk blocking solution, shake by a shaker, and incubate at room temperature for 1 hour.
3. Remove the blocking solution, incubate the membrane in properly diluted primary antibody at room temperature for 2 hours, or at 4? overnight on a shaker.
Note: Dilute the primary antibody with blocking solution at the antibody manufacturer's recommended dilution.
4. Wash the membrane with 4× or 6× TBS-T for 3 times, 10 min each. Increase the volume of wash buffer, and / or the wash times can reduce the background.
Note: Before proceeding Step 4, rinse the membrane with TBS-T simply, the wash efficiency will be increased.
5. Incubate the membrane in properly diluted HRP conjugated secondary antibody at room temperature for 2 hours on a shaker.
Note: Dilute the secondary antibody at the antibody manufacturer's recommended dilution.
6. Repeat Step 4 to remove the non-specific conjugation of HRP conjugated secondary antibody.
Note: Wash the membrane thoroughly after proceeding Step 5.
7. Prepare substrate working solution: mix Reagent A with Reagent B at ratio 1:1, i.e. Mix 1 ml of Reagent A with1 ml of Reagent B, mix thoroughly for use. The dosage of this working solution should be determined according to the membrane size, it is better to immerse the membrane completely in this working solution, i.e. Per 10 cm2 membrane need about 1 ml of this working solution.
8. Put the membrane on a flat (protein side upwards), add the substrate working solution.
9. Incubate the membrane with substrate working solution for 1-5 min.
Note: Observe the incubation in the dark room to determine whether need to do film exposure.
10. Spread a plastic wrap or transparent glassine on the membrane, if the plastic wrap or transparent glassine is big enough, it is unnecessary to remove the excess working solution, but just need slightly press out the bubbles.
11. In the dark room, carefully put a piece of X-ray film on top of the membrane, expose 5 seconds to 1 minute, develop and fix film immediately.
Note: The exposure time can be varied according to chemiluminescence idensity to achieve optimal results. If the signal is weak, prolong the exposure time to hours. Or use a gel visualization or a CCD camera to record chemiluminescent images of the membrane directly.
Preparation and Storage
Store at 4 degree C in dark for one year for frequent use. Or store at -20 degree C for long term.
Related Product Information for Hypersensitive ECL Chemiluminescent Substrate reagent
This substrate is an enhanced chemiluminescent substrate with high sensitivity and unique chemiluminescent system. With low background, good stability, sensitive and enhanced signal, this reagent is good for detecting direct and indirect conjugated HRP antibodies and their related antigens, and can detect the target protein sensitively. Catalyzed by HRP, this chemiluminescent substrate can be used to expose X-ray film, and do luminometer detection directly or fluorescence CCD scan.
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Product Notes
The Hypersensitive ECL Chemiluminescent Substrate (Catalog #AAA355702) is a Reagent and is intended for research purposes only. The product is available for immediate purchase. It is sometimes possible for the material contained within the vial of "Hypersensitive ECL Chemiluminescent Substrate, Reagent" to become dispersed throughout the inside of the vial, particularly around the seal of said vial, during shipment and storage. We always suggest centrifuging these vials to consolidate all of the liquid away from the lid and to the bottom of the vial prior to opening. Please be advised that certain products may require dry ice for shipping and that, if this is the case, an additional dry ice fee may also be required.Precautions
All products in the AAA Biotech catalog are strictly for research-use only, and are absolutely not suitable for use in any sort of medical, therapeutic, prophylactic, in-vivo, or diagnostic capacity. By purchasing a product from AAA Biotech, you are explicitly certifying that said products will be properly tested and used in line with industry standard. AAA Biotech and its authorized distribution partners reserve the right to refuse to fulfill any order if we have any indication that a purchaser may be intending to use a product outside of our accepted criteria.Disclaimer
Though we do strive to guarantee the information represented in this datasheet, AAA Biotech cannot be held responsible for any oversights or imprecisions. AAA Biotech reserves the right to adjust any aspect of this datasheet at any time and without notice. It is the responsibility of the customer to inform AAA Biotech of any product performance issues observed or experienced within 30 days of receipt of said product. To see additional details on this or any of our other policies, please see our Terms & Conditions page.Item has been added to Shopping Cart
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