I have run for detection of IL-10 protein expression on day wise basis. Is this western blot correct?
Is it possible for you to elaborate on your query? There is no marker so how will you determine the band of your interest? Usually, the marker does not appear on the western blot image but it appears on the membrane so for reference we make a tiny cut at the desired band size that helps in analyzing the results of western blotting.
No, I think the western blot in the image is not correct. The IL-10 protein bands are not visible in any of the lanes except the second lane (please specify), which means that the protein is not being expressed in the samples.
There are a few possible reasons for this:
1) The antibody used to detect IL-10 is not specific to the protein
2)The samples were not prepared correctly.
3)The western blot was not performed correctly.
To troubleshoot the problem, you should first check the specificity of the antibody. You can do this by performing a western blot with a control sample that contains known amounts of IL-10 protein. If the antibody does not detect the IL-10 protein in the control sample, then it is not specific and should be replaced.
If the antibody is specific, then you should check the preparation of the samples. Make sure that the samples were properly homogenized and that the protein concentration is high enough to be detected by western blot.
Finally, you should check the performance of the western blot. Make sure that the blot was run at the correct voltage and temperature, and that the transfer was performed correctly.
If you have ruled out all of these possibilities and the western blot is still not producing any bands, then it is possible that IL-10 is not being expressed in the samples. You can confirm this by performing a different assay, such as ELISA or immunohistochemistry.
Thanks and all the best!
Some general techniques you can do to improve your western blots are:
- Run your gels at a lower voltage for longer to get straighter lanes/bands.
- Try a wet transfer if you are finding it hard to detect your protein.
- Try incubating your membrane with the antibody diluted either milk or BSA (some antibodies just image better in either BSA or Milk!)
- Incubate your antibody at 4 degrees overnight to ensure specific binding.
- Try a more sensitive ECL solution if you are struggling to image your protein (signalfire by Cell Signalling is excellent).