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Micro BCA assay: Why can it detect proteins at lower concentrations?
In life science research, protein determination is a crucial part. With the advancement of science, many assay methods have emerged, among which the Micro BCA assay has attracted widespread attention due to its sensitivity to extremely low concentrations of protein. The success of this assay is driven by its unique mechanism and advantages.
Core mechanism of Micro BCA assay
The micro-BCA assay is based on the chemical reaction of bicinchoninic acid (BCA) with copper ions. The reaction first involves the reduction of copper (II) ions to copper (I) ions by peptide bonds in the protein, and then two molecules of BCA bind to each copper (I) ion to form a dark purple complex. This color change is proportional to the concentration of the protein, allowing it to be quantified colorimetrically.
Key to these reactions is the extraordinary sensitivity of the micro-BCA assay to low protein concentrations, particularly in the range of 2 to 40 micrograms per milliliter.
Advantages over traditional methods
The traditional BCA assay is suitable for proteins ranging from 20 to 2000 μg/mL, while the micro-BCA assay can sensitively detect proteins as low as 2 μg/mL. This improvement allows researchers to obtain reliable measurements even when sample concentrations are extremely low.
The greatly improved sensitivity of the micro-BCA assay makes it possible to measure many samples that were previously unmeasurable, which is particularly important for studies involving rare or precious samples.
Experimental steps and precautions
When performing the micro BCA assay, you first need to prepare the corresponding reagents, including micro BCA reagent and copper solution, which can be stored for a long time at room temperature. The key to this process lies in the accurate mixing ratio of reagents and the control of reaction conditions.
For example, Micro BCA Reagent A and B need to be mixed in a ratio of 25:1 and used immediately after preparation to ensure its stability. The samples need to be assayed under appropriate conditions, usually incubating at 37°C to further enhance the sensitivity of the assay.
These details are very important: not only for operational mastery, but also for the overall understanding of the micro-BCA assay.
Application Restrictions
Despite its many advantages, the micro-BCA assay can still be subject to interference in certain circumstances. For example, reducing agents and metal chelators may affect measurement accuracy. Therefore, these substances should be consciously avoided when taking measurements to ensure that the final results are not affected. Especially in biological samples, common membrane lipids and phospholipids may also affect the determination.
SummaryThe development of the micro-BCA assay has not only improved the ability to measure proteins in the micro-concentration range, but also provided a more sensitive and reliable tool for various studies. This not only reflects the accuracy of scientific research, but also provides new perspectives for future biotechnology and medical applications. As technology advances, we can’t help but wonder what innovative methods will emerge in the future to challenge our existing testing standards and limits?
