In Western blotting, sometimes the band obtained for an antibody is not in the region of its MW (specified by the manufacturer) according to the marker. But if we get a single band and used in a fresh membrane, is it OK to use that band for semi-quantitative measurement of that protein?
Before you can make that determination, I would ask a few questions. How far off is your protein band from what is predicted? If it is within 10% than you should be ok. If it is greater than that, there may be issues. I would do the following.
1. Have you tried calling the antibody company and discussing your problem?
2. I would also look at the citations at the bottom of the datasheet and find researchers that are using this antibody for western blot analysis. Look to see where there band was and if it is similar to yours. Also you could ask them if they had any problems with the antibody. A lot of times, proteins will run differently in vivo compared to in vitro situations often present in the datasheets as validation.
3. Do you know if your protein undergoes extensive post-translational modifications which would make it run differently? Could the protein be phosphorylated?
4. Do you have the preabsorbtion peptides to see if the band that you are seeing goes away? If the band goes away, then your band is most likely correct.
I have had similar problems with another antibody for phospho sodium atpase where the band was significantly lower than the predicted size. When I used the preabsorption control the bands went away which led me to believe that I was looking at the proper band. I did further research and found out that if I boiled my samples, I would get a band at 65 kDa and if I did not boil my samples before loading, I would get a band around 100 kDa.
Good luck
Before you can make that determination, I would ask a few questions. How far off is your protein band from what is predicted? If it is within 10% than you should be ok. If it is greater than that, there may be issues. I would do the following.
1. Have you tried calling the antibody company and discussing your problem?
2. I would also look at the citations at the bottom of the datasheet and find researchers that are using this antibody for western blot analysis. Look to see where there band was and if it is similar to yours. Also you could ask them if they had any problems with the antibody. A lot of times, proteins will run differently in vivo compared to in vitro situations often present in the datasheets as validation.
3. Do you know if your protein undergoes extensive post-translational modifications which would make it run differently? Could the protein be phosphorylated?
4. Do you have the preabsorbtion peptides to see if the band that you are seeing goes away? If the band goes away, then your band is most likely correct.
I have had similar problems with another antibody for phospho sodium atpase where the band was significantly lower than the predicted size. When I used the preabsorption control the bands went away which led me to believe that I was looking at the proper band. I did further research and found out that if I boiled my samples, I would get a band at 65 kDa and if I did not boil my samples before loading, I would get a band around 100 kDa.
Good luck
Thanks Dr. Brandon Ambrose Kemp. I was not aware of the preabsorption technique. I will perform preabsorption protocol to identify the specific band.
As Brandon suggested there could be heavy post-translation modification.
Another possibility could be a splice variant in that particular cell lysates.
If you have a negative control, for example lysate which have been knocked down of the protein you are studying, you can see if the band disappears or not.
I agree with the comments by our colleagues.
It would be better if you could show us via email the scan of blotted protein. I also found that the position of the protein band sometimes was different from my expectation. However, we could be sure by investigating the pattern of peptide bands other than the target. I usually add additional nitrocellulose paper below the one to be treated with antigen-antibody reaction, and then stain it with dye (such as Coomassie blue or AgNO3) to know the position of the (whole) peptide including the standard. As a general procedure I also also perform protein staining on the gel after blotting to ensure that the protein has been well transferred. And then I would take care of temperature condition during blotting. If the temperature surrounding the gel and nitrocellulose paper during blotting is not homogeneous and stable, then we could see distorted results. Things like these are trials and errors. I suggest you do it several times. Thank you
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