Protein detection and quantification methods
2023-12-28 07:07:29
Protein detection and quantification methods
Protein quantification methods first appeared in the early 1950s, when physicists and chemists entered the field of biology and applied their techniques to the analysis and measurement of proteins. Lowry protein analysis is the first biochemical analysis to determine the total level of protein in solution. Soon after, the assay was introduced by other measurement methods, including ultraviolet (UV) systems and amino acid analysis. All of these methods are capable of characterizing proteins in water or very mild buffer solutions.
As protein research becomes more complex, quantitative methods begin to develop. In 1976, the Bradford analysis was a stepping stone to study more protein in natural solutions and use detergents or other reducing agents to keep protein soluble. The Lowry test is then re-cultured to the bicinchoninic acid (BCA) assay (also known as the Smith assay), which makes it more suitable for use in solvents for protein research.
However, the latest innovations in protein quantification have existed for more than 25 years. The small series will outline current protein detection and quantification methods and discuss a new infrared-based technology for fast and accurate measurement of trace proteins.
Current status
Some of the most common methods for protein and/or peptide assays include amino acid analysis, ultraviolet (UV) spectroscopy, colorimetric assays, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and densitometry. Immunological based methods and mass spectrometry.
Amino acid analysis
Amino acid analysis measures the amount of each amino acid in a protein and is considered the gold standard method for protein quantification. This method can detect most amino acids with a detection range of 1 mg phenylthiocarbamoyl (PTC) and 5 mg to 10 mg ninhydrin. This method is not as widely used as other assays because it is expensive, slow, and requires technical expertise. Therefore, amino acid analysis laboratories are often used as core facilities.
Ultraviolet spectroscopy
Ultraviolet spectroscopy measures the absorbance of aromatic amino acids in a sample and is the most common method used for protein detection and quantification. Any type of protein can be measured, ranging from 1 mg to 100 mg at 205 nm and 20 mg to 1,000 mg at 280 nm. Ultraviolet spectrophotometers are relatively inexpensive and easy to use.
Colorimetric analysis
A colorimetric assay for protein concentration is determined by a chromogenic reagent. The most common colorimetric assays are dye binding and fluorescence methods. Fluorescent methods include the NanoOrange kit (10 ng to 10 mg at 570 nm) and CBQCA (10 ng to 150 mg at 550 nm), which are generally more sensitive than dye binding methods, including Bradford (1 mg to 50 mg) and Bicinchoninic. . Acid (BCA; 0.2). Magnesium to 50 mg).
Most protein assays are sensitive to solvent conditions. For more information on kits and buffer components, visit the manufacturer's website. This information enables researchers to remove potential interfering substances using protein precipitation or small-scale gel filtration.
SDS-PAGE and density determination
SDS is an anionic detergent that linearizes proteins and covers them with a negative charge. The protein is then separated by size during electrophoresis, and the protein can be measured using quantitative densitometry. For silver staining, the SDS-PAGE protein assay ranged from 1 ng to 5 ng per strip, and for Coomassie Blue, the SDS-PAGE protein assay ranged from 40 ng to 50 ng per strip.
Immunology-based approach
For example, quantitative enzyme-linked immunosorbent assays (ELISA), Western blots, and dot blots are very common and sensitive assays for protein detection and measurement that rely on the use of antibodies for protein capture. The antibodies used in these methods are directed to conformational and linear epitopes. For Western blotting, it is important to use antibodies raised against peptides or denatured proteins because SDS denatures the antigen.
Mass spectrometry
Mass spectrometry is commonly used in functional proteomics to measure small changes in protein abundance in complex biological systems in response to disturbances such as disease progression or drug treatment. However, mass spectrometry can be expensive and slow, and requires specialized knowledge to run the instrument. In addition, the mass spectrometer may not be fully quantified.
Novel detection method
EMD Millipore offers a new protein/peptide quantification method based on a direct detection Fourier transform infrared (FTIR) spectrometer. The system is capable of high precision infrared (IR) measurements of amide bonds in protein chains without relying on amino acid composition, dye binding properties or redox potential.
The protein is represented by 9 amide peaks in the IR spectrum and represents different vibrational modes of the peptide bond. In particular, the amide I and amide II linkages are unique in that their signal strength and purity are the same even in the presence of complex mixtures, reducing agents and detergents. Therefore, these bands are used for measurement. The accuracy of the system is comparable to the accuracy of amino acid analysis
Direct detection systems include protein detection techniques and analytical tools, including BSA standard curves pre-installed on software. This assay requires only 2 microliters of sample, is deposited on a no assay card, placed in the instrument and then measured. The entire process takes about two to three minutes.
Protein detection and quantification are key components of the drug development process. There are many ways to choose from, each with its own strengths and challenges, and there are many factors to consider when choosing the best method for each application.
Protein quantification technology has been in use for more than half a century, but the field has been relatively stagnant for more than 25 years. Developing new tools is critical to providing researchers with fast, easy-to-use methods for protein detection and quantification.
The National Standard Material Resource Platform is mainly engaged in chemical reagents (high-end reagents, general reagents, analytical reagents), standard products, reference materials, experimental chromatographic consumables, instruments and other nearly 200,000 kinds of goods. Another biobw.com agent introduces well-known suppliers at home and abroad, from quality inspection, commodity storage management, professional preservation, logistics and transportation, after-sales service, etc., all the links are controlled by professionals. Through online operation and self-delivery logistics, the 3%-5% of the cost saved will be returned to the customer to ensure the high quality and low price. Imported brands include American ACC, US USP, German DR, German weitge, Canadian TRC, Canadian CDN, BEPURE and other well-known brands, with short delivery time and low price. Click on the online customer service to inquire about the purchase.
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Protein quantification methods first appeared in the early 1950s, when physicists and chemists entered the field of biology and applied their techniques to the analysis and measurement of proteins. Lowry protein analysis is the first biochemical analysis to determine the total level of protein in solution. Soon after, the assay was introduced by other measurement methods, including ultraviolet (UV) systems and amino acid analysis. All of these methods are capable of characterizing proteins in water or very mild buffer solutions.
As protein research becomes more complex, quantitative methods begin to develop. In 1976, the Bradford analysis was a stepping stone to study more protein in natural solutions and use detergents or other reducing agents to keep protein soluble. The Lowry test is then re-cultured to the bicinchoninic acid (BCA) assay (also known as the Smith assay), which makes it more suitable for use in solvents for protein research.
However, the latest innovations in protein quantification have existed for more than 25 years. The small series will outline current protein detection and quantification methods and discuss a new infrared-based technology for fast and accurate measurement of trace proteins.
Current status
Some of the most common methods for protein and/or peptide assays include amino acid analysis, ultraviolet (UV) spectroscopy, colorimetric assays, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and densitometry. Immunological based methods and mass spectrometry.
Amino acid analysis
Amino acid analysis measures the amount of each amino acid in a protein and is considered the gold standard method for protein quantification. This method can detect most amino acids with a detection range of 1 mg phenylthiocarbamoyl (PTC) and 5 mg to 10 mg ninhydrin. This method is not as widely used as other assays because it is expensive, slow, and requires technical expertise. Therefore, amino acid analysis laboratories are often used as core facilities.
Ultraviolet spectroscopy
Ultraviolet spectroscopy measures the absorbance of aromatic amino acids in a sample and is the most common method used for protein detection and quantification. Any type of protein can be measured, ranging from 1 mg to 100 mg at 205 nm and 20 mg to 1,000 mg at 280 nm. Ultraviolet spectrophotometers are relatively inexpensive and easy to use.
Colorimetric analysis
A colorimetric assay for protein concentration is determined by a chromogenic reagent. The most common colorimetric assays are dye binding and fluorescence methods. Fluorescent methods include the NanoOrange kit (10 ng to 10 mg at 570 nm) and CBQCA (10 ng to 150 mg at 550 nm), which are generally more sensitive than dye binding methods, including Bradford (1 mg to 50 mg) and Bicinchoninic. . Acid (BCA; 0.2). Magnesium to 50 mg).
Most protein assays are sensitive to solvent conditions. For more information on kits and buffer components, visit the manufacturer's website. This information enables researchers to remove potential interfering substances using protein precipitation or small-scale gel filtration.
SDS-PAGE and density determination
SDS is an anionic detergent that linearizes proteins and covers them with a negative charge. The protein is then separated by size during electrophoresis, and the protein can be measured using quantitative densitometry. For silver staining, the SDS-PAGE protein assay ranged from 1 ng to 5 ng per strip, and for Coomassie Blue, the SDS-PAGE protein assay ranged from 40 ng to 50 ng per strip.
Immunology-based approach
For example, quantitative enzyme-linked immunosorbent assays (ELISA), Western blots, and dot blots are very common and sensitive assays for protein detection and measurement that rely on the use of antibodies for protein capture. The antibodies used in these methods are directed to conformational and linear epitopes. For Western blotting, it is important to use antibodies raised against peptides or denatured proteins because SDS denatures the antigen.
Mass spectrometry
Mass spectrometry is commonly used in functional proteomics to measure small changes in protein abundance in complex biological systems in response to disturbances such as disease progression or drug treatment. However, mass spectrometry can be expensive and slow, and requires specialized knowledge to run the instrument. In addition, the mass spectrometer may not be fully quantified.
Novel detection method
EMD Millipore offers a new protein/peptide quantification method based on a direct detection Fourier transform infrared (FTIR) spectrometer. The system is capable of high precision infrared (IR) measurements of amide bonds in protein chains without relying on amino acid composition, dye binding properties or redox potential.
The protein is represented by 9 amide peaks in the IR spectrum and represents different vibrational modes of the peptide bond. In particular, the amide I and amide II linkages are unique in that their signal strength and purity are the same even in the presence of complex mixtures, reducing agents and detergents. Therefore, these bands are used for measurement. The accuracy of the system is comparable to the accuracy of amino acid analysis
Direct detection systems include protein detection techniques and analytical tools, including BSA standard curves pre-installed on software. This assay requires only 2 microliters of sample, is deposited on a no assay card, placed in the instrument and then measured. The entire process takes about two to three minutes.
Protein detection and quantification are key components of the drug development process. There are many ways to choose from, each with its own strengths and challenges, and there are many factors to consider when choosing the best method for each application.
Protein quantification technology has been in use for more than half a century, but the field has been relatively stagnant for more than 25 years. Developing new tools is critical to providing researchers with fast, easy-to-use methods for protein detection and quantification.
The National Standard Material Resource Platform is mainly engaged in chemical reagents (high-end reagents, general reagents, analytical reagents), standard products, reference materials, experimental chromatographic consumables, instruments and other nearly 200,000 kinds of goods. Another biobw.com agent introduces well-known suppliers at home and abroad, from quality inspection, commodity storage management, professional preservation, logistics and transportation, after-sales service, etc., all the links are controlled by professionals. Through online operation and self-delivery logistics, the 3%-5% of the cost saved will be returned to the customer to ensure the high quality and low price. Imported brands include American ACC, US USP, German DR, German weitge, Canadian TRC, Canadian CDN, BEPURE and other well-known brands, with short delivery time and low price. Click on the online customer service to inquire about the purchase.
Http://
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