Bovine Serum Albumin (BSA) is a globulin found in bovine serum, containing 607 amino acid residues, with a molecular weight of 66.446 kDa. It plays significant roles in blood circulation, lipid metabolism, detoxification, and has a wide range of applications in biochemical experiments, such as serving as a blocking agent in Western blotting or protecting enzymes from degradation and non-specific adsorption in enzymatic reaction buffers.
When using BSA as a western blot blocking agent, unlike skimmed milk, it cannot be dissolved by vortex shaking. This is primarily due to the fact that vortex stirring may cause denaturation and aggregation of the protein. Vortex stirring may cause perturbation of the protein structure, especially at elevated temperatures, which may lead to the formation of irreversible β-folded structures between the BSA molecules, which can lead to protein aggregation [1].
The main reason why 5% skimmed milk containment solution can be shaken without causing protein denaturation when configured in experiments is that the composition and properties of skimmed milk differ from pure proteins such as BSA in that skimmed milk contains a variety of proteins (e.g., casein and whey proteins) rather than just a single protein. This mixed composition makes it more stable during shaking and resistant to structural damage caused by mechanical agitation. Also, skim milk exhibits some degree of emulsification, which helps to disperse and stabilize the mixture [2].
Theory:
1. Slow addition of buffer solution: Add BSA slowly to a pre-cooled appropriate buffer solution, e.g. by punching along the side wall.
2. Gentle shaking: Shake the container gently to help the BSA dissolve evenly.
3. Standing at room temperature: Let the solution stand at room temperature for a period of time.
4. Avoid excessive concentration: High concentrations of BSA are more difficult to dissolve, and gentle stirring may be required for a long time if a highly concentrated BSA solution is required.
In practice:
We configure 5% BSA: 2.5 grams of BSA in a centrifuge tube, slowly add 50 ml of buffer, put it in the refrigerator at 4 degrees overnight, and it will dissolve the next day.
References
[1] Structural evolution during protein denaturation as induced by different methods. Phys Rev E Stat Nonlin Soft Matter Phys, 2008.
[2] Probing the secondary structure of bovine serum albumin during heat-induced denaturation using mid-infrared fiberoptic sensors. Analyst, 2015.
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