I forgot to add protease inhibitor cocktail before I lysed my tumor tissue samples for protein extraction. For 30 µg of protein I needed an exposure time of 2 seconds for the Actin protein as the loading control. After I forgot the samples overnight at room temperature, I needed 30 seconds of exposure to see any bands from the same samples. (Detection by ECL system) However, I do not observe any smear in the picture, rather sharp bands.
In Western Blot the signals you see come from the antibodies you use for developing your proteins. If your Ab is monoclonal, it is likely to stain only the band that contains the antigenic region of your protein. If the Ab is polyclonal, it might stain several bands. The bands can be nice and sharp because each Ab would only recognize its own specific Ag.
A smear on your gel (or blot) is usually a sign of overloading, not of protein degradation.
If your protein is degraded, you expect to see multiple immunoreactive bands on your blot, just as you did. This can be quite extensive and you may see that many bands that it might look like a smear, but if your resolution is high enough, you should be able to discern the different bands.
Actin is usually very highly expressed in the cells and it is not easy to detect any degradation by WB because high expression usually masks protein degradation.
if you want to test for protein degradation, test for another protein which is not very highly expressed.
if the protein is degraded it may show up at a smaller MW (not usually). Depends if the epitope is still detected.
In Western Blot the signals you see come from the antibodies you use for developing your proteins. If your Ab is monoclonal, it is likely to stain only the band that contains the antigenic region of your protein. If the Ab is polyclonal, it might stain several bands. The bands can be nice and sharp because each Ab would only recognize its own specific Ag.
Thank you everyone for your reply.
I use polyclonal antibodies for my protein detection.
I observed two extra bands at lower molecular weight height in my WB and my inference was that the protein is degraded. However, my seniors denied this. they suggested that usually degraded proteins give a smear.
I doubted this since it was a denaturing gel and so I should not get a smear, rather several bands. I just wanted to confirm if I am right and know if degraded proteins give smear or band in a denaturing gel.
The smear your senior is referring to would be seen in a silver- or Coomasie stained gel, but not in a Western blot. The two smaller bands might be degraded protein. However, if you still do have intact size protein the degration might be partial.
It depends on the levele of degration. It is is partial degradion, you will see a smear bellow the band of interest, and if your protein is highly unstable and fully degraded, then you may see nothing, or bands close to the front.
I agree with Tanja. If you would have ay degradation, you would see a smear band s below your band of interest r nothing sometimes. If you have just smear bands, it is possible that you have no degradation.
At the same time leaving overnight at room temperature is quite risky, I would recommend to repeat it uing the protease cocktail and not leaving overnight and check if you have the same pattern
Dear Gladys Miryam Lopez
we use protease inhibitor cocktail before lysing the tissue in RIPA buffer but during this experiment I forgot to add the cocktail before lysing the tissues and forgot the sample out at RT.
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