eECL Western Blot Kit

Item Number

E665966

• Storage Temp: Store at 2-8°C,Protected from light
• Shipped In: Wet ice

Product Description

Product content

E665966	Component	50 mL	250 mL	Storage
E665966A	eECL-A (Luminol Enhancer)	25 mL	125 mL	2-8℃.Protect from light.
E665966B	eECL-B (Peroxide)	25 mL	125 mL	2-8℃.Protect from light.

Products

The High Sensitivity Chemiluminescent Assay is a highly sensitive and enhanced assay for use with horseradish peroxidase (HRP) in immunoblotting experiments. It is based on a new generation of enhanced chemiluminescent substrate, which is catalyzed by HRP to emit light, and can be used to detect biological macromolecules such as proteins immobilized on membranes. The high sensitivity enables the detection of pg-level antigens with a strong and long-lasting luminescence signal, which can be detected by X-ray film exposure or chemiluminescence imaging.

Caveat

1. Please wear gloves and use clean equipment such as tweezers during the contact with the membrane to avoid protein contamination and high background.

2. Under the condition of avoiding light, the prepared working solution of chemiluminescence detection substrate can be stored stably at room temperature for 8 hours. Sunlight or other strong light may affect the working solution, so avoid prolonged exposure to strong light. Normal laboratory lighting for a short period of time does not affect the use of the working solution.

3. Aladdin Reagent offers a wide range of membranes for protein transfer, containment solutions, primary antibodies, enzyme-labeled secondary antibodies, buffers, etc. For details, please check the company's website.

Procedure

1. After incubation of the secondary antibody, the blotting membrane is washed thoroughly.

2. According to the required amount, eECL-A and eECL-B are mixed in equal volumes at a ratio of 1:1, and prepared as a chemiluminescence detection substrate working solution (about 1m working solution is used for an 8cm×6cm membrane).

3. Discard the washing buffer, add the luminescent substrate working solution dropwise onto the blotting membrane, make sure the working solution covers the whole membrane, and incubate for 3-5 minutes at room temperature.

4. Pipette off excess luminescent substrate working solution and place the blotting membrane between two layers of clean plastic film, this process should be completed carefully to avoid air bubbles between the membrane and the membrane.

5. X-ray film exposure is performed in a dark room, or the membrane is placed inside a chemiluminescence imager and tested according to the instrument instructions.

Schedules

concern	Possible causes	cure
Film reversal (white stripes, black background)	) Excess HRP in the system	Dilute the HRP marker at least 10-fold.
Brown or yellow bands appear on the membrane
Strong glow seen in dark room
Short duration of light signal
Weak or no signal	Excessive HRP in the luminescent reaction system, eliminating Excessive substrate depletion, causing rapid signal reduction	Dilute the HRP marker at least 10-fold.
	Insufficient amount of antigen/antibody	Increased antigen/antibody usage
	Low protein transfer rate	Optimization of the transfer system
high context	Excess HRP in the system	Dilute the HRP marker at least 10-fold.
	insufficiently closed	Optimization of closure procedures
	Poor choice of containment reagents	Select another containment reagent
	Inadequate rinsing	Increase rinsing time, frequency
	overexposure	Reduced exposure time
	High antigen/antibody concentration	Reduce the concentration of antigen/antibody used
Protein bands are punctate	Protein transfer failure	Optimizing the transfer process
	Membrane not in equilibrium	Handle the membrane according to the instructions
	Air bubbles between film and membrane	Remove all air bubbles before exposure
Presence of non-specific bands (high background, short signal duration)	Too much HRP in the system	Dilution of HRP markers
Presence of non-specific bands (background clean signal maintenance time normal)	Excessive use of monoclonal antibody	Further dilution of primary antibody
	SDS leads to non-specific binding	Avoid using SDS during experiments