Protein stabilization is a highly important requirement for in-vitro protein studies alongside serving as major requirements in functional studies. Therefore, it is important to develop an understanding of protein stability alongside the preservation of native conformation. RNA sequencing is also suitable as a genomic approach for detecting and quantitative analysis of messenger RNA molecules in a biological sample. Both of these processes have been crucial contributors to innovation and discovery in medicine in recent times. Researchers worldwide need to understand the common approaches for both techniques to preserve the integrity of lab samples.
Guide on Common Methods for Protein Stabilization
Proteins are constantly subjected to constant flux with specifically defined half-lives. Constant synthesis and degradation of proteins inside the cell for reduction of unnecessary protein load and prevention of undesirable effects are also evident. Furthermore, sudden changes in the native environment can influence the structural and functional properties of proteins.
However, these outcomes can be responsible for precipitation, degradation, or denaturation of proteins, making the protein useless. Therefore, many experimental methods have emerged over the years for the identification and measurement of protein structure and stability.
Some of the common methods for protein stabilization include the following:
Differential Scanning Calorimetry
Differential Scanning Calorimetry or DSC is a common technique implemented to characterize protein stability in the native form. DSC technique involves the measurement of the amount of hearing needed for denaturing a specific protein. It provides better advantages over other methods to determine protein stability, such as thermodynamic parameters and simplicity of sample preparation.
The Pulse-Chase method for determining protein stability relies on labeling cells with pulse or radioactive precursors. It is one of the traditional choices for determining protein stability. Many researchers prefer the pulse-chase method due to the support for accurate measurement of protein’s half-life and identification of subcellular localization. The two most common non-radioactive variants of the pulse-chase method include the cycloheximide-chase method and bleach-chase method.
Fluorescence-Based Activity Assays
Fluorescence-based activity assays are one of the preferred methods for the indirect measurement of protein stability. They can be useful for obtaining information on protein functionality and activity. On the other hand, the susceptibility of the assays to artifacts can create challenges for developing reproducible outcomes. Furthermore, the dyes used for tracking protein stability can present risks for the stability of the concerned protein.
Circular Dichroism Spectroscopy
Circular Dichroism Spectroscopy or CD spectroscopy can provide required information on the stability and conformation of proteins. The approach in CD spectroscopy involves the measurement of differences in absorption between right and left-handed polarized light generating from a structural asymmetry of the concerned protein. CD spectroscopy is the common method for determining protein stability in many laboratories due to the requirement of minimal amounts of sample, easier operation, and non-destructive nature.
Guide on Basic Steps for RNA Sequencing
The basic steps for RNA Sequencing studies are also critical for understanding the best approaches to preserve the integrity of laboratory samples. Many studies for RNA sequencing have pointed out conventional techniques such as emerging developments in computational, wet-lab, or bio-informatics tools. However, the traditional methodological pipeline has been a prominent factor for the efficiency of RNA sequencing studies. Here is an outline of the steps that come under the concerned traditional methodological pipeline,
- Efficient isolation of single, viable cells from the tissues under concern for the study with recommendations of emerging technologies like split-pooling
- Breakdown of isolated individual cells for capturing maximum RNA molecules possible
- Conversion of poly[T]-primed mRNA to complementary DNA by leveraging reverse transcriptase
- Amplification of small amounts of complementary DNA through PCR or in-vitro transcription
- Pooling and sequencing of amplified complementary DNA through NGS by leveraging library preparation techniques, genomic-alignment tools, and sequencing platforms
The gradually emerging trends in biomedical research are transforming the conventional approaches for innovation. The fast-track modifications in biomedical research require advanced solutions that preserve lab samples effectively and improve researchers’ productivity.
Among one of the notable issues for biomedical research, degradation or denaturation of samples always stood first. Furthermore, the impact of physical environments also created various setbacks for efficiency in biomedical research. However, the arrival of new solutions is transforming the usual approaches alongside improving their efficiency considerably. Explore more about such groundbreaking solutions right here now!