No signal of my normaliser, Western blot

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In summary: I haven't experienced that, but I would check that your stripping buffer is calibrated and free of inhibitors (glycine). I would also experiment with different stripping buffers, including ones that are high in salt (such as Tris or NaCl), to see if that resolves the issue.
  • #1
mountain
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Dear everybody,

Please help me out.

I am doing an experiment where i use B actin as the normalizer. The problem is after i have stripped the filters then some of the wells did not have the B actin signals. Even if the intensity of the target protein in that well had the same strength as the others, so this could not be a loading problem, could it? What is wrong? I bought the mouse monoclonal antibody against human B actin from Santa Cruz Biotechnology, Inc. Has anyone used this before? I used 500X dilution. Should i reduce the dilution factor? Do you know any well known manufacturers for selling good antibody against human B actin?

Thanks!
 
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  • #2
I don't know if there's a problem with your beta-actin detection method (anything from a bad antibody to a bad method), a problem with damaging/washing off the beta-actin when you strip the antibody off, or if your samples are truly variable in the amount of beta-actin in them (this is why you need to choose the control carefully for normalizing; you could be loading unevenly or there could be variation in beta actin in your samples, which would indicate it's not a good protein to normalize to in your experiments).

The first place I'd check is whether it's in the stripping step or antibody. Try detecting beta-actin first, before the other protein of interest, and see if you still get the variation. If you do, it's not the stripping but something else, but if you don't have the same problem, then you need to troubleshoot your stripping step.

The other thing you can do, and should have started with, is a dilution series with your antibody to determine the optimal dilution for your system. If you're detecting your protein in some lanes but not others, then I wouldn't suspect the antibody.

Have you confirmed that you're running the gels an appropriate time and with proper acrylamide content to detect both your protein and the beta actin? Could your beta actin still be trapped in the loading gel (especially considering your other post asking about uneven running of gels)?
 
  • #3
what are you using as a stripping buffer? glycine at pH 3?

personally, i would not strip to detect the actin unless you absolutely have to.
 
  • #4
Thanks for the replies.

I have experienced that after stripping then one of my filters lost all of the signals even the MW marker. I wonder if stripping can do this as a side effect? Yesterday i stripped again and then reprobed with B actin and this time i have got the signals back. Have you experienced this?

Thanks again!
 

1. Why am I not getting any signal on my normaliser in my Western blot?

There are several reasons why you might not be getting any signal on your normaliser in your Western blot. One possible reason is that the normaliser protein is not expressed in the samples you are testing. Another possibility is that the normaliser protein is present, but at a very low level, making it difficult to detect. It is also possible that there was an error in the experimental process, such as improper sample preparation or handling, that resulted in the loss of the normaliser protein. Lastly, it is important to consider the sensitivity of your detection method and ensure that it is capable of detecting the normaliser protein at the concentration present in your samples.

2. How can I troubleshoot the lack of signal on my normaliser in my Western blot?

If you are not getting any signal on your normaliser in your Western blot, there are several steps you can take to troubleshoot the issue. First, double check your sample preparation and handling to ensure that the normaliser protein was not lost during the process. Next, try using a different detection method or increasing the sensitivity of your current method to see if the normaliser protein can be detected. It may also be helpful to compare your results to a positive control sample that is known to contain the normaliser protein. If all else fails, consider repeating the experiment with a different batch of samples or normaliser protein to rule out any potential errors.

3. Can I use a different normaliser if I am not getting a signal on my current one in my Western blot?

Yes, it is possible to use a different normaliser if you are not getting a signal on your current one in your Western blot. However, it is important to choose a normaliser that is appropriate for your samples and experimental conditions. This means considering factors such as expression levels, stability, and suitability for the detection method being used. It is also important to note any potential interactions or cross-reactivity that may occur between the new normaliser and other proteins in your samples.

4. Is it possible to have a successful Western blot without a normaliser?

In most cases, it is not recommended to perform a Western blot without a normaliser. The purpose of a normaliser is to control for variations in sample loading and transfer efficiency, ensuring that the results obtained are reliable and accurate. Without a normaliser, it can be difficult to interpret the results and draw meaningful conclusions. However, there may be some situations where a normaliser is not necessary, such as when comparing the expression of a single protein in different samples. In these cases, it is important to carefully consider the experimental design and ensure that appropriate controls are included.

5. How can I prevent the loss of my normaliser protein during the Western blot process?

To prevent the loss of your normaliser protein during the Western blot process, it is important to take proper precautions during sample preparation and handling. This includes using appropriate lysis buffers and protease inhibitors to prevent degradation of the normaliser protein. It is also important to handle the samples gently and avoid repeated freeze-thaw cycles, as these can lead to protein denaturation and loss. Additionally, it may be helpful to use a protein concentration assay to determine the amount of normaliser protein present in the samples before loading them onto the gel, which can help ensure equal loading and minimize loss during transfer.

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