If you have ever worked with Bovine Serum Albumin (BSA), you know that suspending the fluffy crystals in solution can often be a complicated process. BSA is a highly soluble protein due to its strong negative charge and affinity for water. However, there are a number of factors that can impact how quickly and easily the BSA solubilizes. Below is a checklist of Do’s & Don’ts for the best process and methods to apply when suspending BSA.
1. Do learn the science behind BSA suspension
Before determining the best BSA suspension method, it’s important to know the science behind it. The albumin molecule gets its unique globular shape from the folding of amino acids. The secondary structure of amino acids, that form the albumin protein, have areas that are both hydrophilic (high affinity for water) and hydrophobic (repels from water) and each interact differently with the solution (Source: Masuelli, 2013). When BSA is enhanced with exogenous lipids, they take longer to suspend because the lipids bind to the hydrophilic sites. Subsequently, the molecule prevents ions in solution from associating with those sites, thus reducing the protein’s overall interaction with water. Similarly, polymerized albumin molecules bind to other molecules through hydrophilic sites, preventing more frequent interactions with the solution. All of these aspects should be clearly understood as the process moves forward.
2. Do establish a step-by-step process
The best procedure for dissolving BSA is to “wet” the solution by layering it. This should begin by adding a layer of BSA into the mixing vessel followed by the water or buffer solution. Most professionals attempt to layer the BSA on top of the solution, but doing so can actually take longer to suspend because the material will float; and, often, create dust. This is a problem because the more albumin dust generated means more of the BSA is lost and provides opportunities to impact the concentration.
3. Don’t foam the solution
It is not uncommon to see foaming when making a BSA solution, but this can lead to longer suspension times. Foam is brought on by the protein’s dual polymeric and polyelectrolyte nature which causes repulsions, or bubbles, during the absorption process (Krzan, Caps and Vandewalle, 2013.) To avoid foaming, do not agitate or use stir bars to mix the solution. Instead, swirl or rock the solution periodically until there are no remaining crystals or sediment at the bottom of the container. Foaming must be avoided as it can cause trap small lumps of BSA in the foam bubbles which will increase the non-specific binding in the solution.
4. Don’t be limited to hand-mix for large batches.
For small batches of solution, we recommend leaving the BSA to settle on its own with a few occasional swirls. Large batches should be stirred on the lowest setting of the mixing equipment you are using; it may take several days to fully suspend BSA in a large batch. Additionally, if you are adding salts to your solution, adding the salts to the reconstitution process can help the polar portions of the protein disassociate, creating more interactions and therefore allowing it to be more soluble.
5. Don’t store or stir more than necessary
In its lyophilized form, and when prepared in an area with low contamination, the BSA can be stored at room temperature. However, once in solution, the recommended storage temperature is 2-8 degrees C to best prevent microbiological activity. Keep in mind, albumin is more stable in a liquid format and the more it is stirred or agitated, the more interactions it will have causing it to polymerize at a faster rate.
6. Do know the best solutions for suspension.
Solvents are application based, but salt buffers or pure water solutions, will work the best for most applications. Typically, albumin is close to a neutral pH and is soluble up to 35 percent in a salt solution and up to 50 percent in a pure water solution.
7. Don’t choose the wrong size filter for stock solution
A 0.2µm filter is the best choice to reduce the potential for any bacterial contamination. With this filter size, the solution will filter easily unless there are issues with contamination. If it seems difficult to filter through a 0.2µm filter, then a graduated filtration system can help alleviate some issues. For example, starting with a 1µm filter then 0.5µm filter, then ultimately 0.2µm terminal filter. Have all filter sizes available and nearby to keep the process moving forward based on your needs.