I am doing western blots to determine if antibodies are binding selectively to the desired protein in a basal fish. I am using two different antibodies for the same protein in different vertical strips. They have different immunogens. One measures 110 kDa (the predicted molecular mass), the other measures 100 kDa. Uniprot states there is a 100 kDa isoform, but this hasn't been confirmed in our species. Is a 10 kDa difference in molecular mass acceptable considering they were run on different gels and possess different immunogens? What's an acceptable kDa range for the same protein on different gels? Thanks.
You have to adapt the protocol. As you said, the difference is too short, but I think you will be able to separate them with variations in the voltage and other parameters, even in you can bind one of the proteins to other thing (specifically), the little difference could be avoided.
Let me clarify, I am cutting vertical strips on the western blots and hope to see one immunoreactive band of the appropriate size in each lane. I am testing two antibodies for the same protein on separate vertical strips. Antibody A measures about 109 kDa and antibody N measures about 100 kDa. Considering they have different immunogens, is this difference negligible for such a large weight?
The question is whether the measurement of 109 and 100 Da made on separate strips is a statistically significant difference or is within measurement error of being the same mass. One way to address this question is to make the measurements several times, calculate averages and standard deviations, and perform a t-test to see if the difference is significant. Another approach is to combine both antibodies in one blot and see whether you get one band or two, compared with the individual antibodies. This should be done with a light loading of antigen to maximize the chance of seeing two separate bands. Also, you should optimize the separation, if there are two bands, by choosing the most appropriate acrylamide percentage.
It is possible that your protein can have additional epitope in the peptide located between 100K and 109K. In that case your Abs are different. That means that the antibodies have different specificities and you need to consider that.
It's also possible that your protein has some isoforms/glycoforms (precursors) that can have higher MW.
Another possible explanation is that your another antibody just recognizes another protein which has similar epitope.
To test this, you can try to label one of your Ab with radioactive (or some other) label, and see if your two Abs to compete to each other. That would explain if your Abs have the same epitope.
Thank you for your answers. I have BLASTed the genome for sequences that are similar to both epitopes to determine if there are products that the antibodies may improperly bind due to similar sequences. The BLAST searches show only one sequence in the genome that is similar to both epitopes. The same sequence shows up for both immunogen BLAST searches indicating they both may bind to the same product. Also, the epitopes are really well conserved.
Would this genomic evidence, along with a single immunoreactive band in western blots, a Uniprot predicted isoform of 100 kDa and proper immunofluorescent staining be enough convincing evidence for an isoform in a non-model species?
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