What type of BSA protein should I use?
A scientific rationale explaining why fatty acid-free BSA proteins are superior for blocking applications
Bovine serum albumin (BSA) proteins are used by scientists all around the world as a common reagent for coating surfaces to prevent biofouling and to improve biological assay performance. The simple but highly versatile method to use BSA as a blocking agent is relevant to many different areas of scientific research, ranging from laboratory diagnostics to biosensors and nanomedicine.
BSA protein is so widely used because of the broad availability of its source material (i.e., cow blood) and many well-established purification processes. However, there are so many different types of commercially available BSA manufactured through different processes that it can be confusing for scientists who just want to know which one is the best for them. For example, there are more than 40 different types of BSA products on the Sigma-Aldrich website.
Cold ethanol fractionation and heat-shock fractionation are two of the most well-established processes to isolate BSA proteins from cow blood by separating them from other proteins. Due to BSA’s biological role as a fatty acid transporter, BSA proteins contain bound fatty acids that were either naturally bound, or exogenously added as part of the heat-shock fractionation process and can be optionally removed by an extra processing step. Some BSA products are made using this extra processing step and some are not. This leads to two main types of BSA products in the market: fatty acid-free and fatty acid-containing BSA.
From a blocking perspective, there are many fundamental questions that have long been unanswered when it comes to picking the right BSA for the job. Do differently processed BSA proteins possess the same blocking properties? Which BSA type is ideal for blocking applications and why? Does choosing fatty acid-free or fatty acid-containing BSA make any difference for blocking applications? Although there are some online forum discussions about these questions, there are no clear answers and most importantly, until now, essentially no scientific evidence-backed rationale to guide BSA selection for blocking applications.
In our study, we applied a broad range of cutting-edge material science approaches to test six BSA protein options obtained through different purification methods and investigated their conformational and adsorption properties as well as their blocking performance. Fatty acid-containing (“fatted”) BSA proteins purified by cold ethanol fractionation, heat-shock fractionation, or cold ethanol followed by heat-shock fractionation without further purification were designated as BSA 1-3, respectively. An additional fatty acid removal step was performed on BSA 1-3 to yield fatty acid-free (“defatted”) BSA 4-6, respectively.
Solution-phase and adsorption experiments revealed that fatty acid-free BSA proteins had less conformational stability, greater adsorption uptake, and greater surface-induced denaturation on flat and nanostructured glass surfaces than fatty acid-containing BSA proteins. Fatty acid-free BSA proteins also formed superior antifouling coatings when tested in various application settings such as serum biofouling, Western blot, and nanoparticle-related immunological assays.
We confirmed that these performance differences are due to the presence of bound fatty acids which enhance the conformational stability of BSA proteins as well as confer negative charge to the BSA proteins. Consequently, fatty acid-containing BSA proteins do not easily unfold, rendering them less effective surface coatings for blocking applications. In marked contrast, we observed that fatty acid-free BSA proteins form more effective surface coatings because they have less stability and less charge repulsion so they can more easily unfold on a surface and form a densely packed, impenetrable surface coating that is excellent for blocking applications.
We hope that these findings will provide useful evidence-backed guidance to allow scientific researchers to rationally choose the best BSA protein option for their application. For blocking applications, we recommend fatty acid-free proteins.