The Case for Carrier-Free Tau Antibodies
When studying the biology and chemistry of how the tau protein causes disease, there is a lot of nuance to the pathogenesis. The molecular model on which most tauopathies are built involve dissociation from microtubules and aggregation of tau into neurofibrillary tangles (NFTs). However, when we zoom out to actually studying this phenomenon on a cellular level, the formulation of the tools used to study tau becomes very important to hone in on exactly what tau is doing.
Most antibodies are formulated with what we call carrier proteins and bacteriostatic agents [link to formulation blog], which are suitable for broader questions, but when narrowing the scope down to a specific site – like a phospho-site on tau – formulation becomes a key consideration for the quality of quantitation and compatibility with live-cell assays.
The tau protein is highly interconnected to a broad array of signaling pathways, like the PI3K/Akt pathway, MAPK pathways (like p38 and JNK), mTOR signaling, and many more. With 85 putative sites of phosphorylation, discoveries related to tau phosphorylation can be limited by the throughput of an assay. This is why ELISA has become a popular method for quantifying tau phosphorylation as a response to specific stimulus conditions [link to primary research article]. However, the presence of carrier proteins (such as BSA) in ELISA samples can complicate quantification and introduce unnecessary background. Thus, to accurately detect levels of phospho-tau, it is recommended to use carrier-free antibodies such as HUABIO’s PBS-only antibodies so that quantification isn’t masked by unrelated proteins within a sample.
When else could the formulation of a tau antibody obscure important breakthroughs?
Some aspects of tau’s biology require live cells for the monitoring of its consequences, and tau antibodies are valuable for neutralizing tau within a cellular sample. One such case is for the study of a phenomenon known as tau seeding. The term tau seeding essentially refers to the prion-like ability of misfolded tau to transfer its dysregulation from cell-to-cell. Researchers are still working to uncover the mechanisms of tau seeding, which may be influenced by specific phosphorylation sites such as the speculated role for S214 [link to phospho-tau blog].
Some studies have found that tau seeding can be blocked by neutralizing antibodies. As more and more precision work is performed to discover the consequences of each phospho-site, antibody formulation becomes a key consideration. Assays to detect tau seeding rely on live cells, and common bacteriostatic agents like ProClin and sodium azide are highly toxic and may cause cell death unrelated to the biological variables being studied.
Even beyond conjugating tau antibodies for neutralizing applications, having a PBS-only tau antibody ready for bioconjugation finds its application in the world of spatial biology within the realm of multiplexed immunohistochemistry (mIHC). To prepare an mIHC experiment, antibodies are tagged with fluorophores or tags for mass cytometry (such as oligonucleotides). The reactions that target antibodies for bioconjugation are also susceptible to reacting with carrier proteins like BSA, thus resulting in off-target conjugation products that limit the yield of the desired tagged tau antibody. Further, additives like bacteriostatics also limit the efficiency of any conjugation reaction on tau antibodies.
Are PBS-only tau antibodies the only option for high quality tau research?
The short answer is not always. When it comes to selecting the right antibody for an experiment, it comes down to the level of precision needed for the set-up at hand. While PBS-only tau antibodies enable researchers to explore neutralizing antibodies, perform conjugation, and get the most background-free ELISA results, the presence of carriers and bacteriostatic agents is inert to many other experimental workflows. For example, immunohistochemistry (IHC) and western blot (WB) experiments are typically unaffected by primary antibodies that are formulated with these additives. PBS-only tau antibodies may help with background for these applications in theory, but the effect might not be completely evident.
When choosing the materials for your next tau experiment, be sure to consider the formulation. Though the additives in your antibody’s vial may feel inconsequential at times, considering the presence of bacteriostatics and carriers could make a big difference when it comes down to cost and benefit when planning a tight-knit tau experiment.
https://pmc.ncbi.nlm.nih.gov/articles/PMC3924573/#abstract1
https://pubmed.ncbi.nlm.nih.gov/31038156/
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