Product Introduction

Product Name	Cat. No.	Spec.
Hypersensitive ECL Chemiluminescence Kit(Femtogram)	G2020-50ML	2×25 mL
	G2020-100ML	2×50 mL
	G2020-500ML	2×250 mL

Product Description/Introduction

ECL chemiluminescence Kit is a highly sensitive chemiluminescence kit based on luminol ECL chemiluminescence. This product can react with horseradish peroxidase (HRP) conjugated on secondary antibody to produce fluorescence and detect samples by using X-ray lithography or other appropriate fluorescence imaging equipment (fluorescence or chemiluminescence imaging system, etc.). Low concentration samples can still be detected, saving samples. Signal can still be detected after incubation with low concentrations of antibodies (primary, secondary), saving precious antibodies. Signal values are enhanced at least 10-fold compared to the Sensitive ECL Chemiluminescence Kit (G2074).

Storage and Shipping Conditions

Ship with wet ice; Store in the dark at 2-8℃, valid for 12 months.

Product Components

Component Number	Component	G2020-50ML	G2020-100ML	G2020-500ML
G2020-1	Hypersensitive ECL solution A	25 mL	50 mL	250 mL
G2020-2	Hypersensitive ECL solution B	25 mL	50 mL	250 mL
	Product Manual	1 pc

Assay Protocol/Procedures

1. Preparation of hypersensitive ECL working solution: Mix the hypersensitive ECL solution A and hypersensitive ECL solution B in equal volumes, room temperature preparation, ready to use.

2. In Western experiment, PVDF membrane (or NC membrane) is incubated with secondary antibody, washed several times, and excess liquid is absorbed by filter paper. Stick two layers of PE gloves or other transparent films on the exposure box, place the protein side of the PVDF film between the two layers of the exposure box, add the mixed ECL working solution to cover the film and place it on the film for 1-2 min.

3. Remove the ECL working liquid with filter paper or absorbent paper, cover the upper film and start pressing the film.

4. The pressed film should be developed and fixed with developing and fixing reagents (G2019, G2023, G2024 are recommended). Adjust exposure conditions according to luminous intensity.

Note

1. Be sure to change the pipette tips during pipetting of Reagent A and B to avoid cross-contamination, which may result in the failure of active components.

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. Please use up the ECL working solution within one day after it has been configured, do not leave it until the next day, so as not to affect the accuracy of the experimental results.

5. After the use of each solution, please keep the bottle tightly capped and keep it away from light to prevent failure; especially solution B, which contains oxidizing agents and is easily reduced and become ineffective. In addition, liquid A should not be stored close to the inner wall of the refrigerator to prevent the precipitation of crystals due to the local temperature is too low. If crystals are precipitated, dissolve and mix in 37℃ warm water bath before use.

6. ECL chemiluminescence reagent kit selection refer to Table 1, primary antibody and secondary antibody recommended dilution ratio test data are from self-research antibody.

7. Refer to Table 2 for common problems and solutions for ECL chemiluminescent assays.

8. ECL chemiluminescence kit A/B solution are harmful to human body, must be careful when handling, 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 Chemiluminescent Kit Selection Reference Table

Product Name	Normal ECL Chemiluminescence Kit	Sensitive ECL Chemiluminescent Kit	Hypersensitive ECL Chemiluminescence Kit
Cat. No.	G2014	G2074	G2020
Detection limit	Nanogram	Picogram	Femtogram
Recommended dilution ratio of primary antibody (1 mg/mL storage solution)	1:1000-1:5000	1:4000-1:10000	1:10000-30000
Recommended 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, broad applicability	Wide range, broad applicability, high sensitivity, medium-abundance proteins, antibody-sparing	Very high sensitivity, low abundance proteins, low or precious antibody potency

Table2:Common problems and Solutions of ECL Chemiluminescence Detection

Common Problems	Possible Causes	Solutions
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 assays should always be closed with BSA or protein-free closures; the smaller the membrane pore size, the longer the closure and elution time should be
	Inadequate incubation of primary antibody	Appropriately prolonged incubation time (overnight incubation at 4°C)
Weak or No Signal	Low concentration of primary and secondary antibodies used	Increase the concentration of primary and secondary antibodies used
	Low protein abundance	Increase sample volume
	Low protein abundance	Switching to a more sensitive ECL chemiluminescence kit
Rapid fluorescence burst, hollow bands (ghost bands) appear	The fluorescence of the target band is too strong, which rapidly consumes the luminescent substrate, and after the substrate is consumed, it will be anti-white.	Reduce the amount of protein uploaded or the amount of primary and secondary antibodies
Brown or yellow bands appear on the membrane	Over-abundance of HRP in the target region generates large amounts of free radicals, resulting in oxidative inactivation of HRP	Reduce the amount of protein uploaded or the amount of primary and secondary antibodies

Hypersensitive ECL Chemiluminescence Kit (Femtogram)