The Hidden Cause of High Background in Phospho-Tau Western Blots

There is nothing worse than performing a western blot and getting an image with extremely high background. This is especially true for the protein tau, which is typically found in central nervous system tissue, a particularly sensitive and precious sample type. High background can be a result of a variety of factors, but there is one step that all tau researchers can take to control for one specific instance that may be contributing to an excess of background. 

Researchers who investigate tau are after the answers to questions regarding neurodegeneration. The reason why tau is studied in particular for neurodegeneration is because this protein is a master regulator of microtubules. Microtubules are the protein cargo networks of the cell and play an important role in neuronal activity. A major molecular culprit in diseases like Alzheimer’s disease and frontotemporal lobe dementia is that tau becomes dysregulated, dissociates from microtubules, and aggregates into tangles that prevent the nervous tissue cells from performing their homeostatic functions. 

One of the leading models of tau dysregulation originates in its post-translational modification (PTM) status. While tau can be acetylated, glycosylated, and glycated, the key PTM for tau researchers is phosphorylation, a highly dynamic and abundant PTM. In particular, the structural and signaling consequences of phosphorylation in tau have been connected to many areas of neurodegenerative disease pathogenesis. Thus, many researchers target phospho-tau in their studies. However, understanding tau phosphorylation is no easy feat, as there are 85 putative phosphorylation sites on tau. Fortunately, there are many materials available for the study of tau and its different phosphorylated variants. (link out to HUABIO phospho tau products – maybe we make a landing page?)

It is the nuances of studying phosphorylation that can make a huge difference for tau researchers. Many researchers follow the same protocol for western blotting for all types of antibodies: (1) block with non-fat dry milk, (2) incubate primary antibody, (3) wash, (4) incubate secondary antibody, (5) wash, develop, and image. While this overall is fine for any antibody, it is actually the first step that really matters when probing for phospho-tau. 

Why does blocking matter?

The purpose of blocking a membrane during a western blot is to saturate the empty protein-binding spaces on a membrane with a nonspecific protein so that target antibodies do not bind nonspecifically to the membrane. Researchers have adopted non-fat dry milk as a reagent to block membranes as this inexpensive reagent contains a plethora of milk proteins such as casein and whey. By dissolving powdered non-fat dry milk into a buffer containing Tween detergent, one has a cheap and quick way to block however many membranes are on this week's western docket. 

However, the proteins in milk are not always so nonspecific. What this means is that the antibody of choice can potentially crossreact with milk proteins, resulting in decreased detection of the target of interest. While this might sound like little signal should be expected, the milk proteins recognized by the antibody will be dispersed across the membrane, resulting in extreme background across the entire blot. 

This happens particularly when using phospho-specific antibodies. The casein found in non-fat dry milk is highly phosphorylated, and this often leads to nonspecific crossreactivity for antibodies that detect phosphorylated targets. In the case of tau, researchers could be looking at up to 85 different phospho-sites, resulting in heterogeneity in the type of phospho-specific antibody being used and risking crossreactivity with proteins in the blocking reagent.

The solution? Bovine serum albumin (BSA). BSA is actually found in non-fat dry milk, but it is also produced as a lyophilized powder that can easily be dissolved in the same buffers used for non-fat dry milk solution. The advantage of using BSA is that the manufacturing process ensures that BSA contains no phosphorylation, preventing crossreactivity with phospho-specific antibodies used for western blotting, thus circumventing any issues with phospho-specific antibody crossreactivity. 

Does this occur for immunohistochemistry (IHC) as well?

While at times IHC can rely on non-fat dry milk as the blocking reagent,it is far more common to use serum or BSA as a blocking reagent than non-fat dry milk. However,on the off chance that non-fat dry milk is used to block IHC staining of phospho-tau, the result will likewise be nonspecific background. Rather than the dark and oversaturated blots that one would expect from using non-fat dry milk to block a western blot, the staining pattern would show faint, nonspecific signal where phospho-tau antibodies crossreact with the blocking agent.

Not sure which antibody to use for your phospho-tau experiment? Still experiencing background with BSA? Feel free to reach out with technical questions and troubleshooting to our human-monitored support inbox at [email protected].

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View our phospho-tau catalog here.