FAQ
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.
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.
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)
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.
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.
What blocking agents can be used with ECL Western Blotting Detection Reagents?
We recommend the ECL Blocking Agent at 5% dilution in PBST or TBST (0.1% Tween 20) buffer.
We have also found several other blocking agents which work well for ECL Western Blotting on Hybond ECL and Hybond-P membranes. All produced similar sensitivities with only slightly variable signal-to-noise ratios. These chemicals (in PBST) are listed below in descending order of efficacy.
1. 5% Liquid Blocking Reagent for 30 minutes; equivalent or superior results to 5% milk.
2. 3% Bovine Serum Albumin (BSA) for 30 minutes.*
3. 1% Liquid Blocking Reagent + 0.5% BSA for 60 minutes.
4. 2% Fish Gelatin for 60 minutes.
5. 5% Normal Goat Serum for 60 minutes.
Note: BSA is the optimal choice when working with phosphorylated proteins.
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).
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.
How can high background on a Western blot be reduced? |
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.
How can I check whether my ECL reagents are working properly?
You can perform the "blue-light test." Simply combine equal volumes of Detection Reagents 1 and 2 in a microcentrifuge tube. Mix, and add several µL of an HRP-conjugated antibody that is known to be functional to the tube. Mix well and turn out the lights in a dark room. Functional reagents will begin to glow bluish-white fairly rapidly.
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.
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? |
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.
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.