U-Blot® Femto-Sensitive ECL Solution

Product Overview

U-Blot® Femto-Sensitive ECL Solution is a highly sensitive, non-radioactive chemiluminescent substrate based on Luminol for detecting horseradish peroxidase (HRP) immobilized on membranes during immunoblotting, with the ability to detect antigens at low femtogram antigens (ultra-sensitive).

The continuous luminescence lasts up to 8 hours and can be repeatedly exposed to chemiluminescent imaging systems for optimal results, as well as used for stripping immunodetection reagents and reprobing. Its high sensitivity allows greater antibody dilutions (Primary 1:5000-1:100,000; Secondary 1:100,000-1:500,000 (1 mg/ml stock)), significantly reducing antibody consumption.

Specifications

Applications

Used to detect HRP-labeled probes or antibodies in Western Blot, Northern Blot, Southern Blot, Dot Blot.

Compatibility & Usage Notes

Mix the two components in equal volumes (1:1) immediately before use and warm to room temperature. Keep the membrane wet, incubate 3-5 minutes, and avoid excess HRP, which can quench signal.

Safety & Handling

Instructions for Use

• Optimize all steps (loading, gel, transfer, membrane, blocking, antibody concentrations, incubation times) for the best result.
• Perform routine electrophoresis, transfer, HRP-labeled antibody/probe incubation, and washing. Use HRP-labeled detection only.
• During the last wash, freshly prepare working solution by mixing equal volumes of solution A and B; warm to room temperature (use separate tips for A and B).
• Drain the membrane (do not dry) and fully immerse in working solution (100-200 µl/cm²) for 3-5 minutes, then expose. Do not over-incubate.
• Drain excess, place in the imaging device, and expose for seconds to minutes; adjust exposure by signal intensity.

Technical Notes

• Optimize sample amount, antibody dilutions, membrane, and blocking reagent.
• Steps can be done under fluorescent light, but prolonged strong light reduces sensitivity; a darkroom helps.
• Weak bands may need 1-10 h exposure; re-incubate with secondary antibody and re-expose if needed.
• Keep the membrane wet; use a shaker; do not stack membranes in one wash box.
• Use pre-stained markers to size bands; use clean, rust-free, scratch-free tools.
• Working solution is stable ~8 h at room temperature in the dark; sodium azide inhibits HRP.

Troubleshooting

• Brown/yellow bands, short luminous time, whole membrane glows: excess HRP -> dilute HRP-secondary antibody.
• No/weak signal: too much HRP (quenching) -> dilute; poor antibody -> change antibody; transfer failure -> optimize transfer; too little target -> increase load.
• High background: excess HRP -> dilute; incomplete blocking -> replace/extend blocking; incomplete washing -> increase washes; too much antigen/primary antibody -> reduce; low primary-antibody specificity -> change.
• Spots/streaks: incomplete transfer -> optimize; PVDF not fully wetted -> rewet with methanol; bubbles -> remove during exposure; secondary-antibody aggregates -> filter the diluted working solution.
• Non-specific bands: excess HRP -> dilute; poor antibody specificity -> change; SDS-induced binding -> standardize transfer.