Why Tau Antibodies Show Multiple Bands on Western Blots
“This tau antibody is terrible – I am getting so much nonspecific background, and I don’t see anything at my expected molecular weight!” is something a researcher might say after probing for the microtubule-associated protein tau. However, no matter which tau antibody they use, it is actually highly likely that they will see multiple bands when probing for tau. Does that mean that all tau antibodies are low quality? No, it just means that tau is a very special protein.
One of the most important aspects of a western blot result is that the pattern of bands observed matches what is expected for your protein of interest. Typically, one protein should result in a single, crisp, clear band if it was detected successfully. Western blots rely on the separation of proteins via SDS-PAGE, so calculating typically the protein’s molecular weight can tell you where to look for a positive signal during an experiment.
Sometimes, however, mass does not tell the full story alone. SDS-PAGE not only separates proteins based on mass but charge is also a factor.
Tau's functions are highly driven by electrostatic interactions, including its overall net positive character. When probing for tau, its actual molecular weight (expected ~45.8 kD) might not appear exactly where it is expected due to the positive charge making it move more slowly during electrophoresis. Tau has been reported to be detected at molecular weights around 67 kD due to charge-based effects.
Tau’s charge explains the shift in molecular weight, but what are all of these other bands?
The answer lies in alternative splicing. A gene's encoding regions, called exons, are packaged together within a series of non-encoding elements known as introns. This results in a set of “mix-and-match” building blocks that will allow the transcriptional machinery to produce different isoforms of tau. Tau is encoded by the MAPT gene, and the variability arises from exons 2, 3, and 10. Exons 2 and 3 correspond to regions of tau’s N-terminal domain, and exon 10 is located within the microtubule binding domain (MTBD). Different isoforms are better at accessing certain physical spaces within neurons, resulting in a combination of six different tau isoforms being expressed in any one cell.
Tau isoforms are named based on the presence of specific exons, named based on how many repeats in the MTBD (4R or 3R, with exon 10 translated or spliced out, respectively) and how many N-terminal inserts are present, e.g., 2N4R. The molecular weights of the N-terminal inserts are approximately 3 kD each, as is the MTBD repeat that can be spliced out. This creates a range of expected molecular weights for tau spanning from 36.7 kD to 45.8 kD. Because each isoform of tau has a unique molecular weight, it is possible to see up to six different bands when probing for tau with even the most specific antibody.
The range of visible molecular weights increases when considering the charge effect of each spliced exon. This is most drastic for the N-terminal inserts, which occur at the most acidic region of the tau protein. 0N tau migrates much faster, appearing at a molecular weight around 14 kD lower than 2N tau despite only 3 kD actual mass difference. The impact of the presence of the MTBD repeat is much less drastic, inflating the apparent mass difference between 4R and 3R tau by about 2 kD more than the actual 3 kD difference that is calculated for these two isoforms.
So this means we should see six bands every time we probe for tau?
Not exactly. There are a few factors that impact tau banding patterns. Foremost, it is important to check the immunogen for the specific tau antibody at hand if it is located in a region that can be alternatively spliced, only select isoforms will be visible. The developmental stage of the tissue likewise matters – adult brains have all six tau isoforms, but fetal brains only have 0N3R tau. Tissue type also contributes, as the concentration of each isoform varies based on the specific cell type. At most you could see six isoforms, but not all bands will be equally intense, and there are definitely cases where you should expect that single, crisp band that makes a western blot feel good.
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