by Proteins are one of the most important macromolecules present in living organisms. They perform various structural and functional roles in the cell. Due to their critical roles, it is important to accurately quantify and characterize proteins. Protein assays are analytical techniques and procedures used to detect and measure the concentration of proteins in samples. This quantification is necessary in fields like proteomics, biochemistry, molecular biology and biotechnology research.
Common Types of Protein Assays
There are various types of assays categorized based on their working principle. Some of the most commonly used assays are discussed below:
Colorimetric Protein Assays
Colorimetric assays determine protein concentration by measuring absorbance of colored complexes formed as a result of protein reaction with certain reagents. One of the most popular colorimetric assay is Bradford assay. It involves binding of Coomassie Brilliant Blue G-250 dye with protein amino groups and acidic side chains to yield a colored product with intensity proportional to protein concentration. Other colorimetric assays include Bicinchoninic acid (BCA) assay, Lowry assay and Biuret Assay. These are easy to perform and inexpensive methods for routine protein quantification.
Fluorometric Protein Assays
Fluorometric assays determine protein concentration through fluorescence spectroscopy. They rely on increase or decrease in fluorescence intensity upon binding of fluorophore with protein molecules. Fluorescence-based assays include Fluorescein isothiocyanate (FITC) labeling assay and NanoOrange Protein Quantitation kit. These have enhanced sensitivity compared to colorimetric methods.
Western Blotting
Western blotting or immunoblotting aids in detection and qualitative/semi-quantitative analysis of specific proteins separated by electrophoresis. Protein Assays utilizes antibodies against target protein conjugated to enzymes or fluorescent tags for visualization. Besides identification, it helps determine post-translational modifications like glycosylation and phosphorylation.
Mass Spectrometry Based Assays
Analytical techniques like Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) mass spectrometry provide quantitative and qualitative protein identification. They ionize protein samples before analyzing mass-to-charge ratio. Capillary zone electrophoresis coupled to mass spectrometry enables high throughput protein quantitation.
Applications of Assays
Protein quantification is essential for various proteomic studies and research applications like:
– Verification of antibody specificity
– Determining expression levels of target proteins in cell/tissue samples
– Monitoring protein purification yields after chromatography
– Standardization of reagents for immunoprecipitation and gel electrophoresis
– Quantification of protein biomarkers of diseases
– Drug development and pharmacokinetic studies
Accurate protein quantification assays are indispensable for present day proteomic characterization and therapeutic research. Standardized protocols with suitable controls must be followed for reliable protein quantification data. Automation and high throughput techniques are improving proteomic workflows. Proper selection of assay based on sample type, aim and sensitivity requirements is crucial. Upcoming proteomic tools will further expand protein detection capabilities.
Colorimetric and fluorometric assays are most frequently used due to ease of operation. Mass spectrometry and western blotting based methods offer qualitative and targeted analysis capabilities. Improved high throughput protein quantification will drive proteomic discoveries to aid healthcare solutions.
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1. Source: Coherent Market Insights, Public sources, Desk research
2. We have leveraged AI tools to mine information and compile it
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