Amersham ECL Western Blotting Detection Kit
Convenient kit that includes ECL detection reagent, HRP-conjugated antibodies, and blocking agent.
FAQ
Primary antibody detection should be less sensitive compared to primary and secondary antibody detection? Will the signal be as strong when using only labled primary antibodies?
Yes, the signal will be amplified with each probing layer, so if you only have one layer, the signal may be a little lower. So use this detection for targets of medium to high abundance. Avoid it for low abundant targets.
When are stable signals important I only need to detect my signals one time anyway?
Stable signals improve reproducibility and the possibility for you to analyze many blots in parallel. If you for example have 5 blots, it is very hard to react and detect with exactly identical timings. If the signal vary within minutes depending on were in the enzyme (HPR) reaction time curve you are it is impossible to get accurate data. Also, if you want to add blots to your experiment or want to compared with previous data, for example blots from the previous week, it is very helpful with stable signals. If you work with ECL Plus chemifluorescence of ECL Plex fluorescence you will get very stable signals you have the possibility of processing many blots and compare your results between different experiments.
Why is the detection limit lower (4.9 pg compared to 9.8 pg) using film on one slide compared to a previous slide?
It can be explained by experimental variation caused by transfer, dilution series, primary antibody affinity (different lots) etc.
If I label the primary antibody is there not a risk that the binding site is affected?
Yes, the binding affinity of the antibody can be affected if the Dye is conjugated to or close to the binding domain of the antibody. It is important to follow the protocol for antibody labeling so it will be an optimal number of dye molecules incorporated per antibody.
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How can high background on a Western blot be reduced? |
I sometimes have problems with fading signals. What can I do to avoid that?
The ECL reagent has become instable for some reason and the signals fade quickly. It can be caused by contamination of solution A and B or improper storage of the ECL reagent (room temperature).
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How can I reprobe a chemiluminescent ECL Western blot for a different target protein without stripping the blot and starting over at the blocking step? |
What is the definition of limit of detection?
When a protein band has a signal to noise ratio above 3, i.e. the signal is 3 times larger than the variation in background.
Why do I get a negative image on my Western blots? The bands are either white, clear or outlined in black with a low to moderate gray background across the blot.
Excess reporter enzyme (horseradish peroxidase) in a small area can rapidly exhaust its substrate in the short time it takes to move the membrane from the detection reagents to film (or a CCD camera or scanner).
You may be able to visualize your bands by returning the membrane to the detection reagent solutions for 5 – 10 minutes and then repeating the detection. The long-term solution is to reduce the secondary antibody dilution (or whichever antibody is conjugated to the reporter enzyme) or to load less protein onto the gel, thereby decreasing the amount of target.
Is there a problem with incubation of 2 antibodies simultaneously? Is there a risk that the antibodies compete/interfere with one another?
There is a risk if you have epitopes really close together. In most cases this is not a problem. If you know that your epitopes are really close, you can probe 2 blots separately and multiplex with a housekeeping protein instead and then combine the data and get reliable quantitative results.
What is solution A and B, why do you need it?
Solution A contains the substrate molecule (ECL-luminol, ECL Plus-Lumigen) and solution B contains an enhancer, which activates the substrate and the reaction with HRP. It is important to make a fresh mix and not to contaminate the 2 solutions for best performance of the reagent.
How can I optimize my antibody dilutions in an easy way?
Traditionally the dot blot method is used for determining the optimum dilution of antibodies. A more reliable method that mimics a true experiment is to prepare a Western blot and divide it into strips/sections. This takes longer time than dot blots, but more parameters, such as specific signal intensity, background and amount of unspecific detection can be monitored.
By using this method you will be able to chose:
- Optimal dilutions of primary and secondary antibodies
- Optimal sample load
- Optimal blocking agent
- Best species (e.g. rabbit or mouse) of primary antibody
- Best quality of primary antibody (choice of best supplier)
Stripping and re-probing of membranes is time consuming and the results are uncertain if we have lost proteins unevenly across the blot during the stripping procedure. What can I do to avoid this if I have 2 targets of the same size?
If you work with chemiluminescence, you can probe 2 blots in parallel with the same samples loaded. One blot is probed for 1 target + housekeeping protein, the other blot for the second target + housekeeping protein. The normalized signals for target 1 and 2 can be compared if the blots were reacted and detected simultaneously.
For ECL Plex, you can perform multiplex detection of the targets simultaneously and detect the 2 targets separately in different channels. For normalization to house-keeping protein you can perform triplex detection.
Troubleshooting
Find solutions to product related issues. For unlisted issues please contact local Cytiva service representation.
ECL Western blotting detection system
| Possible cause | Suggested remedy |
|---|---|
Incorrect storage of the ECL detection reagents may cause a loss of signal. |
Check the instructions for storage. |
ECL detection reagents may have become contaminated. |
Replace the detection reagents. |
Target protein degradation may occur if the blots are stored incorrectly. |
Optimize storage. |
Some antigens may be affected by the treatments required for electrophoresis. |
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Check protein transfer by staining the gel and/or membrane. |
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Check that transfer equipment is working properly and that the correct procedure has been followed. |
| Possible cause | Suggested remedy |
|---|---|
Too much protein was loaded onto the gel. |
Optimization is required. |
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Electrophoresis and transfer protocols may need optimization. |
The concentrations of primary and secondary antibodies could be too high |
Optimization is required. |
| Possible cause | Suggested remedy |
|---|---|
Incorrect handling can lead to contamination on the blots and/or membrane damage, which may cause non-specific signal. |
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Areas of the blot may have dried during some of the incubations. |
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Blotting technique requires optimization. |
| Possible cause | Suggested remedy |
|---|---|
The membranes were blocked for an insufficient time. |
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There was insufficient Tween in the post antibody washes. |
Optimization is required. |
Insufficient changes of post antibody washes were used. |
Optimization is required. |
The film detection of the signal was allowed to over expose. |
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The level of signal is so high that the film has become completely overloaded. |
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The concentrations of primary and secondary antibodies could be too high. |
Optimization is required. |
Contamination can be transferred to the blots from electrophoresis and related equipment used in blot preparation. |
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Transfer and incubation buffers may have become contaminated. |
Buffers require replacing. |
The blocking agent used was not freshly prepared, was too dilute or was incompatible with the application. |
Try to prepare new blocking agent. |
The level of Tween used in the blocking agent was not sufficient for the application performed. |
Optimization is required. |
The membrane was allowed to dry during some of the incubations |
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The type of membrane used was not compatible with nonradioactive systems. |
Try other membrane. |
The post antibody washes were not performed for a sufficient period of time or were not performed in a high enough volume. |
Optimization is required. |
| Possible cause | Suggested remedy |
|---|---|
Probably bacterial contamination either in reagents, buffers, or protein samples, caused endogeneous peroxidases in bacteria. |
To remove from protein samples, soak blot in 15% H2O2 for 30 minutes, followed by a quick rinse. |
| Possible cause | Suggested remedy |
|---|---|
Negative bands generally occur when protein target is in excess and antibody concentrations are too high. The effect is caused by substrate depletion. |
To rectify this either, reduce the amount of target loaded, use lower antibody concentrations or a combination of both. |
| Possible cause | Suggested remedy |
|---|---|
Film exposure time may have been too short. |
Try to prolong the exposure time. |
The concentration of primary and secondary antibodies could be too low. |
Optimization is required. |
Insufficient protein was loaded on to the gel. |
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Transfer efficiency may have been poor. |
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