ECL Chemiluminescent Substrate Reagent Combo Pack, 200 mL
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SKU : G2161-200ML
หมวดหมู่ : Biochemicals , 1. Chemical and Reagents , Buffers , Servicebio ,
แบรนด์ : Servicebio
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Product Information
Product Name | Cat. No. | Spec. |
ECL Chemiluminescent Substrate Reagent Combo Pack | G2161-200ML | ECL Chemiluminescent Substrate Reagent Kit: 2×50 mL |
Sensitive ECL Chemiluminescent Substrate Reagent Kit: 2×25 mL | ||
Hypersensitive ECL Luminescence Substrate Reagent Kit: 2×25 mL |
Product Descrition/Introduction
This product is composed of three kinds of ECL chemiluminescent substrate reagent with different sensitivities: ECL chemiluminescent substrate reagent Kit, sensitive ECL chemiluminescent substrate reagent Kit, and hypersensitive ECL chemiluminescent substrate reagent Kit. The basic principle of ECL chemiluminescent substrate reagent is based on the chemiluminescence of luminol; it can react with horseradish peroxidase (HRP) coupled to the secondary antibody and emit light, which can be detected by X-ray film exposure or other imaging methods (e.g., fluorescence or chemiluminescence imaging equipment); the detection limits are nanogram, picogram, and femtogram, respectively, which can meet the needs of exposure experiments for different target proteins. Sensitive and hypersensitive ECL chemiluminescent substrate reagent can still detect signals at low sample concentrations, saving samples, and can still detect signals after incubation with the same low concentration of antibodies (primary and secondary), saving precious antibodies; the signal value of Sensitive ECL Chemiluminescent Substrate Reagent Kit is enhanced at least 10 times compared with ECL Chemiluminescent Substrate Reagent Kit, and the signal value of Hypersensitive ECL Luminescence Substrate Reagent Kit is enhanced at least 10 times compared with Sensitive ECL Chemiluminescent Substrate Reagent Kit.
Storage and Shipping Conditions
Ship with wet ice; Store at 2-8 away from light, valid for 12 months.
Product Components
Component Number | Component | G2161-200ML | |
G2161-1 | ECL Chemiluminescent Substrate Reagent kit | ECL reagent A | 50 mL |
G2161-2 | ECL reagent B | 50 mL | |
G2161-3 | Sensitive ECL Chemiluminescent Substrate Reagent kit | Sensitive ECL reagent A | 25 mL |
G2161-4 | Sensitive ECL reagent B | 25 mL | |
G2161-5 | Hypersensitive ECL Luminescence Substrate Reagent kit | Hypersensitive ECL reagent A | 25 mL |
G2161-6 | Hypersensitive ECL reagent B | 25 mL | |
Manual | 1 pc | ||
Assay Protocol/Procedures
1. Mix equal volumes of ECL A and ECL B of the same sensitivity to make the corresponding sensitivity of the ECL working solution at room temperature prior to use.
2. In Western blot experiment, PVDF membrane (or NC membrane) is incubated with secondary antibody, washed several times, and excess liquid is absorbed by filter paper. Place the membrane between two pieces of clean cling film (or PE gloves) and add ECL working solution to cover the surface of the membrane. This process should be done carefully to avoid air bubbles between the cling film and the PVDF membrane (or NC membrane).
3. After sufficient reaction, the excess ECL working solution was removed by filter paper or blotting paper, followed by either a pressurized plate or a fluorescence imager.
4. The pressed film is developed and fixed with developing and fixing reagents (G2019 is recommended). Adjust the exposure conditions according to the luminous intensity.
Note
1. The pipette tips must be replaced during the liquid transferring process of ECL reagent A and ECL reagent B. Cross contamination of reagent A and reagent B will lead to the gradual failure.
2. Please wear gloves and use clean equipment such as tweezers to avoid contamination by exogenous proteins and metal ions during contact with the membrane.
3. Sodium azide inhibits HRP activity and all related reagents should be avoided.
4. In order to avoid affecting the accuracy of the results, please use up the ECL working solution within one day, do not leave it until the next day.
5. Please close the bottle tightly after use and keep it away from light to prevent failure; especially reagent B, which contains oxidants and is easy to be reduced and become ineffective.
6. ECL chemiluminescence substrate reagent kit selection refer to Table 1, primary antibody and secondary antibody recommended dilution ratio test data are from self-research antibody
7. ECL chemiluminescent assay common problems and solutions refer to Table II.
8. ECL reagent A and B are harmful to human body, pay attention to effective protection, avoid direct contact with the human body or inhalation of the respiratory tract.
9. For your safety and health, please wear safety glasses, gloves, or protective clothing.
Table 1: ECL chemiluminescence substrate reagent kit selection reference table
Product Name | ECL chemiluminescence substrate reagent kit | Sensitive ECL chemiluminescence substrate reagent kit | Hypersensitive ECL chemiluminescence substrate reagent kit |
Cat. No. | G2014 | G2074 | G2020 |
Detection limit | Nanogram level | Picogram level | Femtogram level |
Recommened dilution ratio of primary antibody (1 mg/mL storage solution) | 1:1000-1:5000 | 1:4000-1:10000 | 1:10000-30000 |
Recommened dilution ratio of secondary antibody (1 mg/mL storage solution) | 1:1000-1:6000 | 1:3000-1:60000 | 1:6000-150000 |
Characteristics and applicability | Wide range and applicability | Wide range and applicability, high sensitivity, medium abundance protein, saving antibody | Extremely sensitive, low abundance of protein, low or precious antibody titer |
Table 2: Common problems and Solutions of ECL Chemiluminescence Detection
Common Problem | Possible Cause | Possible Solution |
High Background（High background or no specific bands） | Primary and secondary antibody dilution without using the correct buffer and concentration | Appropriately reduce the dilution ratio, reduce the concentration of primary and secondary antibodies |
Blocking time or washing time is too short, or blocking solution is incorrect | Phosphorylated protein detection must be blocked with BSA or protein-free blocking solution. The smaller the membrane pore size, the longer the blocking and elution time | |
The primary antibody is not fully incubated. | Properly extend the incubation time (incubate overnight at 4) | |
Weak or No Signal | Primary and/or secondary antibody concentration too low | Increase the concentration of primary and secondary antibodies |
Low protein abundance | Increase the sample volume | |
Switch to ECL chemiluminescence kit with higher sensitivity | ||
The fluorescence is quenched rapidly and a hollow band (ghost band) appears. | The fluorescence of the target band is too strong, which consumes the luminous substrate quickly. After the substrate is consumed, it will appear to be white. | Reduce the amount of protein or the amount of primary and secondary antibodies. |
Brown or yellow bands appear on the membrane | The content of HRP enzyme in the target area is too rich, which produces a large number of free radicals, resulting in the oxidation inactivation of HRP enzyme. | Reduce the amount of protein samples or the amount of primary and secondary antibodies. |
For Research Use Only!
Version: v1.0-202308
