Description
Femto ECL substrate kit for Western blot
GenieBlot Femto ECL Substrate Kit is a two-component, maximum-sensitivity ECL HRP substrate for detecting low-femtogram amounts of protein on Western blots. It is supplied as separate peroxide and luminol/enhancer solutions for fresh mixing, making it ideal for low-abundance targets and limited samples.
The luminol-based substrate is oxidised in the presence of horseradish peroxidase (HRP) and peroxide, giving femtogram-level antigen detection with a detection threshold of 32 fg. NC or PVDF membranes incubated with the working solution can be imaged on X-ray film or a CCD imaging system. The kit suits blot detection, such as Western blot and EMSA, with HRP-labelled antibodies or nucleic acid probes. Signals resist overexposure and bands remain intact after 4 h. For research use only.
Key Features
| High sensitivity | Detects as little as 32 fg of protein. | |
| Resists overexposure | High-microgram protein bands take 2 min to overexpose. | |
| Short exposure | Automatic exposure times of hundreds of milliseconds. | |
| Long-lasting signal | Intact bands can still be observed after 4 h. |
Specifications
| Detection method | Luminol-based enhanced chemiluminescence (HRP substrate) |
| Format | Two components (Substrate A and Peroxide B), mixed 1:1 |
| Detection threshold | 32 fg protein |
| Applications | Western blot, EMSA and other blots with HRP-labelled antibodies or nucleic acid probes |
| Membranes | NC, PVDF |
| Imaging | X-ray film or CCD imaging system |
| Recommended volume | About 70 μl/cm² membrane |
| Shelf life | 2 years |
Components
| Components | 100 ml | 500 ml |
| Femto ECL Substrate A | 50 ml | 250 ml |
| Femto ECL Peroxide B | 50 ml | 250 ml |
Storage
Store at 2 ~ 8°C.
- Wash the blot
- Perform the Western blot as required, then wash off the secondary antibody with PBST/PBS or TBST/TBS.
- Prepare the ECL working solution
- Mix Femto ECL Substrate A and Femto ECL Peroxide B 1:1 immediately before use.
- Use about 70 μl/cm² membrane; adjust to suit your workflow.
- Incubate the membrane
- Lift the membrane with flat tweezers and drain the wash solution on filter paper. Do not let the membrane dry completely.
- Add the working solution evenly to cover the membrane and incubate at room temperature for 1 min.
- Remove excess substrate
- Hold the membrane vertically on filter paper for a few seconds to drain excess working solution.
- 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 tape the edges. Choose the exposure time according to signal development, or take several exposures of different lengths.
- CCD imaging system: plastic wrap is not needed. Follow the imager's instructions.
Q1: What antibody dilutions should I start with?
- For most antibodies, 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: How long is the working solution usable?
- Use it immediately after mixing. It can still be used after several hours at room temperature, with a slight decrease in sensitivity.
Q3: Which membranes and imaging systems are compatible?
- NC and PVDF membranes can be used, and blots can be imaged on X-ray film or a CCD imaging system.
Q4: Can I use sodium azide in my buffers?
- No. Sodium azide (NaN3) inhibits HRP and should be kept out of all buffers.
