Hi everyone
I am working with a 60kDa protein (according to the datasheet), in a 10% SDS gel. The membrane shows 3 sets of double bands, and the one in the middle is faded compared to the other two. The first one is next/above the 75kDa mark, and the last one is right on the 50kDa mark (the last part of the double band is under 50kDa). According to the datasheet, I should analyze the set of the middle; however, I can't help myself stop thinking about analyzing the last set, since is obviously more marked (like the first set) and is nearly the molecular weight that supposed to be (not exactly, like the faded set, but nearly). So, summing up my question, what is more important, the molecular weight or the marking intensity?
Hello Aimée Obolari Durço
In a denaturing western, the protein is denatured to its primary structure and separated by size with smaller molecules moving more quickly through the matrix. So, if you know the molecular weight of your protein of interest, you may confirm the presence of the target protein in the sample by running the protein molecular weight marker and comparing the size of the target protein with the marker.
Only after you confirm the presence of the protein band of interest, you may want to analyze the intensity of the band. The signal intensity of the band is directly proportional to the concentration of the target protein. By analyzing the intensity of the signal, you may determine whether the expression of your target protein in one sample has increased or decreased relative to another sample or the control.
The presence of many bands in Western Blot could be because of the following reasons:
1. Incomplete blocking will cause primary and secondary antibody to bind to the membrane or anything else other than the protein of interest.
2. Sometimes due to poor handling of the sample prior to Western Blot analysis can result in multiple bands due to degradation of sample.
3. If excessive amount of lysate is loaded on the gel antibody binds non-specifically to proteins which is in excess resulting in multiple bands.
4. Sometimes target protein may form multimers which can result in high molecular weight bands than the predicted molecular weight of target protein.
5. If protein is cleaved post-translationally and if one uses polyclonal antibody then multiple bands will be observed because of the ability of polyclonal Ab to recognize multiple epitope.
6. Alternate splicing of mRNA can result in multiple protein products. If the epitope which the antibody recognizes is shared between the proteins then multiple bands can be observed.
7. Post-translational modification of target protein like glycosylation, phosphorylation, acetylation or methylation can result in high molecular weight bands.
8. Lastly, if the antibody is contaminated or unpurified it can result in multiple bands.
So, the band which you would wish to analyze as shown in the blot (2) just because it is more marked is not the proper way since it is not the band of interest, as the molecular weight of your target protein is 60kDa.
Therefore, the molecular weight becomes important as it will help you to identify the protein band of interest with the help of the protein molecular weight marker.
Best.
thank you so much Malcolm Nobre
it really helped me
best regards
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