Are you familiar with the importance of immunoassays? These tests are of crucial value for determining the existence of antigens and antibodies in the blood of individuals.
Since proteins play an indispensable role in detecting antibodies and antigens, it’s no wonder these are used in immunoassays. Nevertheless, determining protein stability is a complicated procedure, which has to be performed under specific conditions in order to provide accurate results. Nowadays, there are multiple assay methods, each of which offers unique benefits.
Learn more about every technique in detail.
DSC (Differential Scanning Calorimetry)
One of the most popular methods for determining protein stability is Differential Scanning Calorimetry. In order to alter the natural qualities of a certain biomolecule, the technique uses heat measurement. Biomolecules with low thermal transition midpoints are believed to lack the required stability unlike those with high midpoints.
In addition, the accurate results provided by DSC have made this analysis the most prominent when compared to other similar techniques. Hence, it’s largely used in choosing the best proteins for the creation of bio-therapeutic medicines. These drugs are based on the use of living organisms, which make protein modifications in order to deliver new treatments.
Differential Scanning Calorimetry is believed to offer a vast array of benefits. The purpose of a protein stability assay is to determine the presence of a particular disease. Proteins are essential in the detection of antibodies and antigens, thus used in immunoassays. DSC enables a simple process of sample preparation, as well as the use of thermodynamics in the determination of parameters.
Moreover, the samples used during the examination can be both solid and liquid, which isn’t the case in many other methods. Denaturation temperatures can also be measured by using Differential Scanning Calorimetry.
The pulse-chase technique used for the purpose of determining protein stability has been popular for years in spite of the use of radioactive amino acids. It involves two stages of cellular process examination in a given time period. In the course of the first stage, cells are under exposure to a labeled compound, known as pulse. During the second stage, cells are exposed to the unlabeled form of the identical compound, known as the chase.
The ultimate goal of the pulse-chase method is to detect the activity of given cells by monitoring their condition in a particular timeframe. It has proven surprisingly accurate in determining the half-life of proteins.
Due to the employment of radioactive amino acids, scientists have come up with non-radioactive versions of the same technique. Anyhow, these have never reached the popularity of the pulse-chase method, including the bleach-chase method and the cycloheximide-chase assay. See this website to learn about the mechanism and uses of a pulse-chase analysis.
The bleach-chase technique involves using fluorophore and a light pulse in the course of bleaching. As far as the cycloheximide-chase method is concerned, cycloheximide is used instead of radioactive amino acids for the purpose of preventing synthesis.
CD (Circular Dichroism) spectroscopy
CD spectroscopy is widely popular in laboratories in spite of the type of information it provides. Lab technicians prefer this technique due to the small samples it requires for determining the stability of proteins.
Additionally, the information provided by this analysis is based on the asymmetry of their structure. By detecting the absorption differences caused by structural asymmetry, CD spectroscopy offers reliable results, which lack a bit of resolution.
Fluorescence activity assays
Last but not least, activity assays based on fluorescence are well-known in the field of protein stability assays. Unlike the other types of analysis, this one determines the stability in an indirect manner. Nevertheless, it provides useful information about the activity and functionality of biomolecules. Check out the following link, https://www.britannica.com/science/fluorescence, for a better understanding of fluorescence.
Nevertheless, the fluorescence activity assay isn’t always reliable in the results it provides. The use of dyes in the process of analysis has a major impact on the stability of proteins. Hence, the outcome of the analysis might not be what researchers hope for.
The bottom line
Regardless of the technique lab technicians use, the ultimate goal is for the stability rate to be accurate.
It’s the only way for immunoassays to prove reliable!