FAQ - ReadyTector®
Method of single-step immunodetection
Why does ReadyTector® accelerate immunodetection?
With ReadyTector®, blocking and immunodetection are carried out simultaneously. ReadyTector® blocks and incubates the blotted membrane with the primary and secondary antibody complexes in one step. This is only possible due to the fact that ReadyTector® can block the membrane much faster and more effectively compared to standard blockers. Therefore the primary and secondary antibodies don’t bind to the membrane non-specifically. There is no non-specific binding and background. With the classic Western blotting protocol this has to be prevented by several sequential washing and incubation steps. With ReadyTector® you can save several working and washing steps compared to the classic method.
Is the ReadyTector® solution with my valuable primary antibody re-usable?
Yes. The ReadyTector® solution with the primary antibody is re-usable up to 5 times without great loss in quality. The detection is always fast and the background will stay low. With every use, the concentration of the primary antibody decreases and so the bands may be weaker. This can be adjusted by minimally longer exposition on X-ray film or imager. More excessive use is not recommended due to consumption of blocking reagent and secondary antibody. This will lead to poor and unreproducible results. The good quality of ReadyTector® will be lost. You can save solution and primary antibody by re-using the solution with the antibodies up to 5 times. Additional ReadyTector® Wash Buffer can be purchased separately.
Do I always have to use 20 mL per membrane as indicated in the instructions?
The volumes for washing and detection indicated in the instructions refer to the dimensions of the membrane for a minigel (10 x 8 cm). For other dimensions the volumes have to be adjusted. If you use small incubation vessels or tubes, the volumes should not be significantly reduced due to the risk of high background.
Which detection methods are compatible with ReadyTector®?
ReadyTector® with HRP labeled secondary antibody can be used with most detection methods such as ECL substrates, X-ray films or imagers. ReadyTector® is compatible with these common detection methods. Detection with TMB substrates is also possible, if the membrane contains a larger amount of protein.
Do I need additional chemicals or equipment for the use of ReadyTector®?
ReadyTector® can be easily integrated into the classical Western blotting procedure. The gel running and the blotting steps remain unchanged. The treatment of the membrane and the following immunodetection will be simplified and accelerated because blocking and immunodetection with primary and secondary antibodies are carried out in one step. Detection can be improved by using the ReadyTector® Chemiluminescent Substrate. Using other substrates is also possible but can lead to higher background and more spots.
Which membrane can I use?
ReadyTector® works with all common membranes such as PVDF and nitrocellulose. Also membranes for “Turbo” or Fast blotters work without any difficulty. We extensively tested different membranes of several producers and didn’t find any membrane that failed.
What does ReadyTector® contain?
ReadyTector® is ready to use and contains components necessary for a one-step immunodetection. You only have to add your specific primary antibody. All other reagents such as blockers or secondary antibody conjugates are already included.
Why is there a special ReadyTector® Chemiluminescent Substrate?
ReadyTector® Anti-Mouse-HRP can be used with all common HRP substrates. It works with TMB and ECL substrates of all tested manufacturers. During our tests we observed background and spots with many ECL substrates although correct immunodetection and blocking took place. Such spots occur independently whether using the sequential protocol or the faster one step immunodetection protocol. Therefore we optimized the ReadyTector® Chemiluminescent Substrate to minimize background and spots. Other very expensive “femto” substrates do not show better detection limits than the ReadyTector® Chemiluminescent Substrate. The ReadyTector® Chemiluminescent Substrate is all-purpose for any protein quantity.
What does a ReadyTector® Kit contain?
The ReadyTector® kits include the detection solution with the secondary antibody (e.g. Anti-Mouse HRP labeled) and the 10 x Wash Buffer. If you re-use the ReadyTector® solution several times you will need more Wash Buffer. The 10x Wash Buffer can be purchased separately (order number 700 500). In addition you need a substrate. For ECL detection we recommend the ReadyTector® Chemiluminescent Substrate due to reduced background and spots. This substrate can be used for high as well as extremely low amounts of protein. But in principle other ECL or TMB substrates can also be used, if a slightly higher background or spots are not a problem.
Quality of results
Does ReadyTector® diminish the results due to fast immunodetection?
No. In most cases ReadyTector® can prevent non-specific binding of the antibodies to the membrane. Binding of the secondary antibodies to the primary antibodies is very fast and efficient. Therefore you can achieve very good results without background notwithstanding this accelerated method. But this all depends on the characteristics of your primary antibody.
What does the background look like if I change to ReadyTector®?
In most cases the background is quite low, comparable with the standard method (if the standard method was optimized carefully). The background also depends on the membrane and the primary antibodies. If you observe background, please increase the incubation time from 1 hour to 2 hours. Normally the background will disappear.
What do I have to consider for obtaining low background?
ReadyTector® enables very good results. However it is recommendable to use all components of the ReadyTector® system together. The use of alternative Wash Buffers as well as alternative substrates can lead to bad results, higher background, more spots and lower detection limits in our tests. Such other components are not optimized and feasible for the new and fast all-in-one detection.
First testing and change to ReadyTector®
How can I test the compatibility of my primary antibody with ReadyTector®?
For an initial testing you can divide the membrane into two identical halves. One half can be detected with the standard method. This half works as a control if there is protein blotted on the membrane and if the primary antibody works. The other half can be used for immunodetection with ReadyTector®. The primary antibody should be used in the initial testing at a maximum concentration (0.5µg/mL). After detection of the two membrane halves you can compare the results of the tested primary antibodies. If you are not sure about the affinity of your primary antibody, please increase the incubation time in your first testing from 1 hour to 2 hours. If there is no signal visible after 2 hours incubation in ReadyTector®, your primary antibody is not a good candidate for ReadyTector®. One reason could be the affinity, which strongly influences the speed of the binding. With most primary antibodies the signal will be very good. If so, you can reduce the incubation time for most primaries to 1 hour and the results will still be fine. Therefore you can take full advantage of the speed of ReadyTector® (If you have very good primary antibodies, incubation times of 15 minutes may be sufficient). Also the concentration of the primary antibodies can be adjusted (reduced) within the concentration range (0.2-0.5µg/mL). If in the first testing there is no band visible after 2 hours incubation with the primary antibody, it could be, that the concentration of the primary antibody is too low or that the primary antibody is not applicable to the fast one step immunodetection.
There is only a pictorial manual in the package? Are the written instructions missing?
No. The flow diagram is the whole manual. The use of ReadyTector® is so easy that you don’t need more detailed instructions.
- Wash the blotted membrane 3 times with the ReadyTector® Wash Buffer
- Add the primary antibody to the ReadyTector® solution and incubate ~1 hour (for high affinity antibodies, incubation time can be reduced)
- Wash the membrane 3-4 times with the ReadyTector® Wash Buffer
The membrane is ready for detection
Please find more information about how to use ReadyTector® at https://www.youtube.com/watch?v=hdjz6A2lC6o
I do not know the concentration of my primary antibody. In the instructions it states that a concentration of 0.2 -0.5 µg/mL should be added. What can I do?
Many manufacturers do not indicate a concentration of their antibodies. But normally they indicate a dilution factor for using these antibodies in Western blotting (e.g. 1:5000). For using these antibodies with ReadyTector® you can follow these instructions. In our testing, these suggested dilutions worked perfectly with ReadyTector®.
Can ReadyTector® be used with any primary antibody?
Basically, yes. ReadyTector® was successfully tested with a high number of different primary antibodies. However, the primary antibody should bind the target protein specifically and with high affinity. To clarify the usability of your specific primary antibody, we recommend testing to learn about the binding characteristics of your specific primary antibody. With adequate primary antibodies, detection limits identical to standard immunodetection protocols are possible. Simply faster and easier. ReadyTector® can only work with primary antibodies of the correct species. ReadyTector® Anti-Mouse-HRP can only be used with mouse primary antibodies and not with primary antibodies derived from rabbit or from other species.
Are there any antibodies which do not work with ReadyTector®?
Yes. The fast ReadyTector® immunodetection needs antibodies with high affinity. Most antibodies show this high affinity. But there are also many commercially available antibodies showing very low affinity. Such antibodies will show a white membrane or no bands after using ReadyTector®. In this case it could be possible to obtain bands after 2 hours of incubation. Otherwise you should detect with the old-fashioned sequential protocol. For such low-affinity primary antibodies, we recommend incubation of the primary antibody overnight to give the antibody enough time to bind.
Does a purchased primary antibody always work very well with ReadyTector®?
No. There are commercial antibodies which have poor affinity. Antibodies with poor affinity often bind other proteins and produce false bands. To ensure good antibody binding you should select an antibody which is compatible with ReadyTector®. Good antibody quality helps to improve the results or in short: “garbage in … garbage out.” We recommend selecting an antibody with high affinity and specificity before starting your tests. Your results will never be better than your primary antibody.
Which criteria were used for selecting the secondary antibody in ReadyTector®?
ReadyTector® contains a labeled secondary antibody, optimized for one-step immunodetection. This antibody conjugate binds very rapidly and gives good and stable substrate turnover. In many tests with different primary antibodies, this selected secondary antibody has stood the test very well.
Antibodies of different species are used for Western blotting. ReadyTector® is only available as Anti-Mouse and Anti-Rabbit so far. Are there solutions for other species?
Further species are in the pipeline. If you are interested in other species, please do not hesitate to contact us.
Washing and Wash Buffer
Why do I have to use a special Wash Buffer with ReadyTector®?
The ReadyTector® solution and the ReadyTector® Wash Buffer are the perfect combination. Washing of the membrane before detection and washing before adding the substrate are important steps during the fast one-step immunodetection. You could also use other washing buffers but this will provide inferior results with standard washing buffers. There will be more background and the strength of the bands will decrease. This is due to the fact that the fast immunodetection needs a different preparation for the membrane, which is done by the ReadyTector® Wash Buffer. Thus we only recommend the ReadyTector® 10x Wash Buffer. The Buffer can be purchased separately in 500 mL bottles.
Why is it important to wash with the ReadyTector® Wash Buffer?
The first washing step after blotting is necessary to remove the components of the transfer buffer (e.g. methanol or ethanol) from the membrane. The ReadyTector® Wash Buffer prepares the membrane for the extremely fast blocking step and prevents nonspecific binding of the primary and secondary antibody to the membrane. The preparation of the membrane is important for reproducible and good results. Otherwise many spots, background or smears can occur on the membrane. The preparation of the membrane for the fast one step incubation is important so using other Wash Buffers will not help. The washing step before adding the substrate is also important to avoid background.
How often do I have to wash after incubation with the antibodies - 3 times or 4 times?
After incubation with the antibody containing ReadyTector® solution you need a washing step to remove unbound (primary and secondary) antibodies to avoid background. If there is visible background, it will be better to wash 4 times. But it depends on the primary antibody. If you get background with a primary antibody, you should always wash 4 times. In most cases the proposed 3 x washing step before adding the substrate is sufficient.