Description
Pico ECL substrate kit for Western blot
GenieBlot Pico ECL Substrate Kit is a two-component enhanced chemiluminescent (ECL) HRP substrate for routine Western blot detection at picogram sensitivity. It is supplied as separate substrate and peroxide solutions for fresh 1:1 mixing, and delivers bright, durable signal compatible with film and digital imagers.
The luminol-based substrate is oxidised in the presence of HRP and peroxide, producing prolonged chemiluminescence that allows picogram-level antigen detection. Membranes (e.g. NC or PVDF) incubated with the working solution can be imaged on X-ray film or with a CCD imaging system. The kit resists overexposure and keeps bands visible for hours. It is suitable for blot detection, such as Western blot and EMSA, using HRP-labelled antibodies or nucleic acid probes. For research use only.
Key Features
| High sensitivity | Detects protein down to the picogram level. | |
| Resists overexposure | High-microgram protein bands tolerate exposure times of up to 15 min. | |
| Long-lasting signal | Intact bands can still be observed after 6 h. | |
| Flexible imaging | Compatible with X-ray film and CCD imaging systems. |
Specifications
| Technology | Luminol-based enhanced chemiluminescence (HRP substrate) |
| Format | Two components, mixed 1:1 before use |
| Sensitivity | Picogram level |
| Applications | Western blot, EMSA and other blots with HRP-labelled antibodies or nucleic acid probes |
| Working volume | About 70 μl/cm2 membrane |
| Detection | X-ray film or CCD imaging system |
| Shelf life | 2 years |
Components
| Components | 100 ml | 500 ml |
| Pico ECL Substrate A | 50 ml | 250 ml |
| Pico ECL Peroxide B | 50 ml | 250 ml |
Storage
Store at 2 ~ 8°C.
- Wash the membrane
- Perform the Western blot as required, then wash off the secondary antibody with PBST/PBS or TBST/TBS.
- Prepare the ECL working solution
- Mix Pico ECL Substrate A and Pico ECL Peroxide B 1:1 immediately before use.
- Use about 70 μl of working solution per cm2 of membrane (adjust as preferred).
- Incubate the membrane
- Lift the membrane with flat tweezers and drain the wash solution on filter paper without letting the membrane dry completely.
- Add the working solution evenly so the membrane is fully covered, and incubate at room temperature for 1 min.
- Remove excess substrate
- Hold the membrane vertically with flat tweezers on filter paper for a few seconds to drain excess working solution.
- For film imaging, quickly place the blot between two layers of plastic wrap and press out any bubbles.
- Image the blot
- X-ray film: place the membrane protein side up in the film cassette and secure the edges with tape. Choose the exposure time from the signal development, or take several exposures of different lengths for the best result.
- CCD imaging system: no plastic wrap is needed; follow the imager's instructions.
Q1: How much working solution do I need?
- About 70 μl of working solution per cm2 of membrane, prepared by mixing Pico ECL Substrate A and Pico ECL Peroxide B 1:1. You can adjust the volume to suit your workflow.
Q2: How long is the working solution stable?
- Use it immediately after preparation. It can still be used after several hours at room temperature, with a slight decrease in sensitivity.
Q3: Can I use sodium azide in my buffers?
- Avoid it, or keep it at ≤0.01%, because sodium azide inhibits HRP.
Q4: What should I use for very low-abundance proteins?
- For low-abundance targets, the GenieBlot Femto ECL Substrate Kit is recommended.
Q5: Why is my background high or my bands unclear?
- Adjust the exposure time to the target abundance: underexposure makes bands unclear and overexposure darkens the background. Poor-quality plastic wrap can also contaminate the membrane and cause high background or signal quenching.
