All-in-One Solution for Western Blotting
1. How does ReadyTector® accelerate immune detection?
With the use of ReadyTector® in Western blotting, blocking and immunodetection are carried out at the same time. ReadyTector® blocks and brings primary and secondary antibodies to the blotted membrane in one step. This is only possible because ReadyTector® blocks the membrane in a much faster and more effective way than is possible with classic blockers. Therefore, the primary and secondary antibodies do not “stick” nonspecifically to the membrane, although they hit the membrane at the same time as the blockage. There is no nonspecific binding and background that have to be laboriously prevented in the classic process through the various incubations and multiple washing steps. This saves a number of work and washing steps in the classic process.
2. Can I use the ReadyTector® solution multiple times with my expensive primary antibody?
Yes. The ReadyTector® solution can be reused up to five times with the primary antibody five times without compromising the quality of the results. The detection is always just as fast and the background remains low. Of course, the primary antibody slowly dilutes with each use, so that the bands may become somewhat weaker. However, this can easily be compensated for by a minimally longer exposure time on the X-ray film or the chemiluminescence detector. However, it is not recommended to use it more than 5 times, because the blocking reagents and the secondary antibodies are also used up. There are then unsightly and possibly not reproducible results.
3. Does the volume of 20ml per membrane, as stated in the package insert, always have to be adhered to?
The volume for washing and for detection in the package insert refers to the membrane size for a mini gel, i.e. about 10 x 8 cm. With other membrane sizes, you should convert accordingly to the current area and use less or more solution. The volumes should not be significantly reduced, even when using smaller incubation vessels or when using tubes for incubation, because otherwise too few of the components of the solutions can act on the membrane.
4. Which detection methods are compatible with ReadyTector®?
ReadyTector® with HRP-labeled secondary antibodies can be used with various ECL substrates. These ECL substrates can be read out on X-ray film as well as with chemiluminescence imagers. ReadyTector® is compatible with these common detection methods. Detection with TMB substrates is also possible, provided the amount of protein on the membrane is large enough.
5. Do you need additional chemicals or additional equipment when using ReadyTector®?
ReadyTector® is simply built into the classic Western blotting process. The protein separation and blotting steps remain unchanged. The treatment of the membrane up to the finished immunodetection is significantly simplified and accelerated by ReadyTector®, since blocking and immunodetection with primary and secondary antibodies take place at the same time. The detection can be improved with the ReadyTector® Chemiluminescent Substrate, but it can also be carried out with any other substrates. However, other substrates generally show an increased background or more spots in the background.
6. Which membrane can I use for ReadyTector®?
ReadyTector® works with all common membrane types, both with PVDF and with nitrocellulose membranes. Membranes for fast blotters (so-called turbo or fast blotters) also work without any problems with ReadyTector®. Various membranes from many manufacturers were tested in extensive tests. So far, no membrane has been found that causes problems.
7. What does ReadyTector® contain?
ReadyTector® is ready-to-use and contains all the components required for one-step immunodetection. All you have to do is add your specific primary antibody. All other reagents such as blockers and secondary antibody conjugate are already included.
8. Why is there a special ReadyTector® chemiluminescent substrate?
ReadyTector® Anti-Mouse HRP can in principle be used with all common HRP substrates. It works with TMB substrates and with the ECL substrates of all manufacturers tested. During the testing, however, it was noticed that many ECL substrates show background and sometimes make various spots on the membrane, although the immunodetection and the blocking functioned perfectly. Such spots can occur regardless of whether the traditional method or the rapid one-step immunodetection are used sequentially. For this reason, the ReadyTector® Chemiluminescent Substrate has been optimized so that there is even less background and, above all, the spots are reduced. In addition, very expensive "Femto" substrates did not show any better detection limits in our tests than the ReadyTector® Chemiluminescent Substrate. The ReadyTector® Chemiluminescent Substrate can therefore be used universally for any amount of protein.
9. What is in a ReadyTector® Kit?
The ReadyTector® kits contain the detection solution with the secondary antibody (e.g. Anti-Mouse labeled with HRP) and a bottle of 10x ReadyTector® washing buffer. If you want to use the detection solution with your own primary antibody several times, you also need additional ReadyTector® washing buffer and a substrate. For ECL detection, we recommend the ReadyTector® Chemiluminescent Substrate. In combination with the new and fast one-step immunodetection, it shows significantly less background and fewer spots than established substrates from other manufacturers. It can also be used universally for both high and extremely low amounts of protein. But in principle, other ECL or TMB substrates also work.
10. Does the faster immune detection with ReadyTector® make the result worse?
No. ReadyTector® is able to largely prevent nonspecific binding of the antibodies to the membrane. The binding of the secondary antibody to the primary antibody is very good and fast. This means that, despite the acceleration of the process by ReadyTector®, a very good result without a background is achieved.
11. What about the background when the method is switched to ReadyTector®?
In most cases, the background with ReadyTector® is less or just as small as with the classic method (if this has been well optimized). However, the background depends, among other things, on the membranes and the primary antibody used. If there is background, increase the incubation time with ReadyTector® from one hour to two hours. Usually there is no longer a background.
12. Do I have to pay attention to something in order to get as little background as possible?
Working with ReadyTector® enables very good results. However, it makes sense to use all components of the ReadyTector® system together. Both the use of alternative washing buffers and the use of alternative substrates have repeatedly shown poorer results in tests with a higher background, more spots, or a poorer detection limit. Understandably, other components are not optimized for the new all-in-one detection at high speed and therefore do not work as well here.
13. The ReadyTector® pack does not come with any written instructions, only an illustrated flow chart. Is the manual missing?
No. The diagram is the whole guide. ReadyTector® is so easy to use that no further instructions are required. The blotted membrane is washed three times. The primary antibody is added to the ReadyTector® solution and incubated for about 1 hour (possibly shorter if the primary antibody has a high affinity) and then washed 3 to 4 times with the ReadyTector® washing buffer. The membrane is then ready for detection with a substrate.
14. Can ReadyTector® be used with any primary antibody?
Generally, yes. ReadyTector® has already been used successfully with a large number of different primary antibodies. However, the primary antibody should bind the target protein specifically and with good affinity. Since the manufacturer's specifications for antibodies with regard to the actual binding behavior are usually rather unclear, testing with your special primary antibody is the only way to clarify the question of suitability with certainty. Identical detection limits can be achieved with suitable primary antibodies and ReadyTector®, as is possible with classic multi-stage immunodetection. ReadyTector® can only work if you use a primary antibody from the “right species”. ReadyTector® Anti-Mouse HRP can be used, for example, with primary antibodies from the mouse.
15. Are there primary antibodies with which ReadyTector® does not work at all?
Yes. Rapid ReadyTector® immunodetection requires primary antibodies with a good affinity. Most antibodies show such a good affinity. However, there are also many antibodies - including commercial ones from a wide variety of suppliers - that show only a very low affinity. With ReadyTector®, such antibodies simply show a white membrane without the desired band. In such a case, with ReadyTector®, you may still be able to see the bands after a 2-hour incubation. Otherwise, one simply has to detect in the traditional sequential way. It is then advisable to incubate the primary antibody overnight because it definitely takes a long time to bind well.
16. What criteria was used to select the secondary antibody contained in the ReadyTector® solution?
The ReadyTector® solution contains a labeled secondary antibody that is ideal for one-step immunodetection. This antibody conjugate binds particularly quickly and provides good and stable substrate conversion. The selected secondary antibody has proven itself in many tests with different primary antibodies.
17. Why does a special washing solution have to be used with ReadyTector®?
The ReadyTector® solution and the ReadyTector® wash buffer are optimally matched to one another. In the case of rapid one-step immunodetection, both washing the membrane before detection and washing before adding the substrate are very important steps. You can use other washing buffers, but then the results will be worse with more background and decreased band strength. We therefore only recommend the use of the ReadyTector® wash buffer. It is available individually as a 10-fold stock solution in 500ml bottles.
18. Why is washing with the ReadyTector® Wash Buffer so important?
The first washing step after the blotting serves, among other things, to ensure that the components of the transfer buffer, such as methanol or ethanol, are reliably washed out of the membrane. In addition, the ReadyTector® washing buffer prepares the membrane for the extremely fast blocking and thus reliably prevents nonspecific binding of the primary and secondary antibodies to the membrane. This preparation of the membrane is important for reproducibly good results. Otherwise, there may be streaks, many spots, or a flat background on the membrane.
19. How often do I have to wash 3 or 4 times after incubation with the antibodies?
After incubation with the ReadyTector® incubation solution with the antibodies, washing is necessary to wash away unbound antibodies (primary and secondary) in order to avoid background. It is sometimes better to wash 4 times if the background is otherwise visible. This depends on the primary antibody. If you get a background with a primary antibody, then you should always wash 4 times with this antibody. However, the suggested 3 washing steps is usually sufficient before adding the substrate.
