Description
Ready-to-use Femto ECL substrate for Western blot
GenieBlot Femto ECL Ready-to-Use Substrate is a premixed, single-bottle, maximum-sensitivity ECL HRP substrate for low-femtogram protein detection on Western blots. It is ready to use straight from the bottle, combining single-solution convenience with the sensitivity needed for low-abundance targets.
This luminol-based enhanced chemiluminescent substrate detects horseradish peroxidase (HRP): HRP and peroxide oxidise luminol, enabling femtogram detection of antigen. Unlike conventional two-part ECL kits, there is no need to mix equal volumes of solution A and B, which avoids reagent loss and cross-contamination during pipetting and improves consistency and reproducibility. The signal lasts long enough for imaging up to 5 hours. Membranes (e.g. NC, PVDF) can be imaged on X-ray film or a CCD imaging system. It suits blotting detection (e.g. Western blot, EMSA) with HRP-labelled antibodies or nucleic acid probes. For research use only.
Key Features
| Ready to use | Single-component format needs no mixing and minimises contamination. | |
| High sensitivity | Detects proteins at low femtogram (fg) levels. | |
| Long signal duration | Imaging is possible for up to 5 hours. | |
| Reproducible results | No A/B mixing step, so less reagent loss and better consistency. |
Specifications
| Detection chemistry | Luminol-based enhanced chemiluminescence (HRP substrate) |
| Format | Single-component, ready-to-use solution (light yellow, transparent) |
| Sensitivity | Low femtogram (fg) level |
| Signal duration | Imaging possible for up to 5 hours |
| Applications | Western blot, EMSA and other blots using HRP-labelled antibodies or nucleic acid probes |
| Compatible membranes | NC, PVDF |
| Imaging | X-ray film or CCD imaging system |
Components
| Components | 100 ml | 500 ml |
| GenieBlot Femto ECL Ready-to-Use Substrate | 100 ml | 2 × 250 ml |
Storage
Store at 2 ~ 8°C.
- Wash the membrane
- Perform the Western blot as usual, then wash off the secondary antibody with PBST/PBS or TBST/TBS.
- Apply the substrate
- Lift the membrane with flat tweezers and drain the wash solution onto filter paper. Do not let the membrane dry completely.
- Drop the substrate evenly onto the membrane to cover it uniformly (about 70 μl/cm2 of membrane; adjust as preferred).
- Incubate at room temperature for 1 min.
- Remove excess substrate
- Hold the membrane vertically on filter paper with flat tweezers for a few seconds.
- Wrap the blot
- 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 according to signal development, or take several exposures of different lengths.
- CCD imaging system: no plastic wrap is needed; image according to the imager's instructions.
Q1: What antibody dilutions should I start with?
- Because the substrate is highly sensitive, start at 1:1,000–1:4,000 for the primary antibody and 1:5,000–1:10,000 for the secondary antibody. Antibody concentrations that are too high can cause high background or white bands.
Q2: Do I need to mix two solutions before use?
- No. It is a single-component, ready-to-use reagent, which avoids reagent loss and cross-contamination and improves reproducibility.
Q3: Can I use sodium azide in my buffers?
- No. Sodium azide (NaN3) inhibits HRP and should not be used in buffers.
Q4: How long can I image the blot?
- The signal allows imaging for up to 5 hours, on X-ray film or a CCD imaging system.
