FAQ
How can I reprobe a chemiluminescent ECL Advance Western blot for a different target protein without stripping the blot and starting over at the blocking step?
You can easily reprobe an HRP-detected Western blot for a protein of significantly different size than the original target (if the bands for the two proteins would overlap, you should consider stripping the blot and reprobing instead).
After initial detection using ECL advance chemiluminescent system, the reagents can be rinsed off with 2 x 10 minute washes in PBS-T ( PBS + 0.3% TWEEN 20). The membrane is then incubated for 30 minutes in 15% H2O2 in PBS to inactivate the horseradish peroxidase enzyme.
Wash the blot 3 x 5 minutes in PBS-T, and cover with the ECL advance reagents for one minute. Wrap the blot in Saran Wrap and expose it to Hyperfilm ECL for 1 minute. A blank exposure indicates that all of the HRP has been inactivated. If you can still see black bands, repeat the PBS-T rinse/hydrogen peroxide/rinse/detection steps.
Once you have inactivated all of the HRP, rinse off the detection reagents with 2 x 10 minute PBS-T washes. You may now probe the membrane with a new primary antibody; follow the detection method's protocol for the rest of the immunodetection.
Remember that this procedure is for reprobing a chemiluminescent Western blot for a new target protein of significantly different size than the original. If you have a chemifluorescent blot, if the second target is the same size as the first, or you are trying to reprobe for the same protein, you should strip the blot according to the detection method's procedure prior to blocking and primary antibody incubation.
How can I reprobe a chemiluminescent ECL Plus Western blot for a different target protein without stripping the blot and starting over at the blocking step? |
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)
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.
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.
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.
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.
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.
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.
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).