💧Mastering Biomolecule Measurements: DNA, RNA, and Protein Quantification with Nanodrop
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3 سال پیش
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NANODROP Spectrophotometry is a full
NANODROP Spectrophotometry is a full spectrum (220nm-720nm) spectrophotometer and can be used to measure concentration of Proteins (A280nm), Nucleic acids (DNA/RNA: A260nm) and assess their purity (salt and organic compound contaminants at A230nm).
Protein absorbance at 280nm directly measures tryptophan and tyrosine amino acids as a surrogate read for total protein content. The tryptophan and tyrosine content of many proteins remains fairly constant, and so the absorbance of protein solutions at 280nm is a good surrogate. The advantages of this method are that the procedure is simple to carry out, it is nondestructive, and no special reagents are required. The major disadvantage is that nucleic acids also absorb strongly at 280 nm and could therefore interfere with the measurement of the protein if they are present in sufficient concentrations. This is why you need to know the 260/280 ratio.
2ul sample vol. is recommended but you could use 1.5ul.
Can have dynamic range of 0.1 – 400 mg/mL
Absorbance at 260nm measures nucleic acids (different nucleotides absorb light at different wavelengths, but average absorption at 260nm is good indicator).
Absorbance at 230nm shows if contaminates such as carbohydrates, phenol or salts (e.g EDTA) and other organic reagents are present. This is relevant for nucleic acid purity checks.
Caveats for protein quantitation:
The Protein A280 method is applicable to purified proteins that contain Trp, Tyr residues or Cys-Cys disulphide bonds and exhibit absorbance at 280 nm.
Colorimetric assays such as BCA, Pierce 660 nm, Bradford, and Lowry require standard curves and are more commonly used for uncharacterized protein solutions and cell lysates.
The bicinchoninic acid assay (BCA assay), also known as the Smith assay, after its inventor, Paul K. Smith measures total proteins (0.5 μg/mL to 1.5 mg/mL) in a solution. You then measure absorbance not at 280nm where interfering biomelecules may also absorb. peptide bonds in protein are reduced by Cu2+ ions from the copper(II) sulfate to Cu+. The amount of Cu2+ reduced is proportional to the amount of protein present in the solution. Next, two molecules of bicinchoninic acid chelate with each Cu+ ion, forming a purple-colored complex that strongly absorbs light at a wavelength of 562 nm. So You are also detecting peptide bonds rather than relying on just typtophan and tyrosine light absorption.
Lowry Method - named for the biochemist Oliver H. Lowry who developed the reagent in the 1940s. His 1951 paper describing the technique is the most-highly cited paper ever in the scientific literature, cited over 300,000 times.
The Lowry method combines the biuret reagent - which detects peptide bonds - with another reagent (the Folin-Ciocalteau phenol reagent) which reacts with tyrosine and tryptophan residues in proteins. This gives a bluish color which can be read somewhere between 500 - 750 nm depending on the sensitivity required. There is a small peak around 500 nm that can be used to determine high protein concentrations and a large peak around 750 nm that can be used to determine low protein concentrations. This method is more sensitive to low concentrations of proteins than the biuret method.
reaction is strongly influenced by four amino acid residues (cysteine, cystine, tyrosine, and tryptophan) in the amino acid sequence of the protein.
Bradford protein assay was developed by Marion M. Bradford in 1976: a colorimetric protein assay, is based on an absorbance shift of the dye Coomassie Brilliant Blue G-250.
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This video is about protein A260/280 ratio, nanodrop data interpretation, nanodrop results meaning, Nanodrop A260/280, Nanodrop protein concentration, Nanodrop dna quantification, nanodrop spectrophotometer, a260/a280 ratio rna, a260/a280 ratio higher than 2, a260/a280 nanodrop, a260/a280 value of pure rna, a260/a280 value of pure dna, Nanodrop spectrophotometry, Biomolecule measurements, DNA quantification
RNA quantification, Protein quantification, UV-Vis spectroscopy, Nucleic acid concentration, Absorbance spectroscopy, Spectrophotometric analysis
Biomolecular assays, Quantitative analysis methods, Molecular biology techniques, Laboratory instrumentation, Nanodrop spectrophotometer tutorial and Precise biomolecule quantitation.
Protein absorbance at 280nm directly measures tryptophan and tyrosine amino acids as a surrogate read for total protein content. The tryptophan and tyrosine content of many proteins remains fairly constant, and so the absorbance of protein solutions at 280nm is a good surrogate. The advantages of this method are that the procedure is simple to carry out, it is nondestructive, and no special reagents are required. The major disadvantage is that nucleic acids also absorb strongly at 280 nm and could therefore interfere with the measurement of the protein if they are present in sufficient concentrations. This is why you need to know the 260/280 ratio.
2ul sample vol. is recommended but you could use 1.5ul.
Can have dynamic range of 0.1 – 400 mg/mL
Absorbance at 260nm measures nucleic acids (different nucleotides absorb light at different wavelengths, but average absorption at 260nm is good indicator).
Absorbance at 230nm shows if contaminates such as carbohydrates, phenol or salts (e.g EDTA) and other organic reagents are present. This is relevant for nucleic acid purity checks.
Caveats for protein quantitation:
The Protein A280 method is applicable to purified proteins that contain Trp, Tyr residues or Cys-Cys disulphide bonds and exhibit absorbance at 280 nm.
Colorimetric assays such as BCA, Pierce 660 nm, Bradford, and Lowry require standard curves and are more commonly used for uncharacterized protein solutions and cell lysates.
The bicinchoninic acid assay (BCA assay), also known as the Smith assay, after its inventor, Paul K. Smith measures total proteins (0.5 μg/mL to 1.5 mg/mL) in a solution. You then measure absorbance not at 280nm where interfering biomelecules may also absorb. peptide bonds in protein are reduced by Cu2+ ions from the copper(II) sulfate to Cu+. The amount of Cu2+ reduced is proportional to the amount of protein present in the solution. Next, two molecules of bicinchoninic acid chelate with each Cu+ ion, forming a purple-colored complex that strongly absorbs light at a wavelength of 562 nm. So You are also detecting peptide bonds rather than relying on just typtophan and tyrosine light absorption.
Lowry Method - named for the biochemist Oliver H. Lowry who developed the reagent in the 1940s. His 1951 paper describing the technique is the most-highly cited paper ever in the scientific literature, cited over 300,000 times.
The Lowry method combines the biuret reagent - which detects peptide bonds - with another reagent (the Folin-Ciocalteau phenol reagent) which reacts with tyrosine and tryptophan residues in proteins. This gives a bluish color which can be read somewhere between 500 - 750 nm depending on the sensitivity required. There is a small peak around 500 nm that can be used to determine high protein concentrations and a large peak around 750 nm that can be used to determine low protein concentrations. This method is more sensitive to low concentrations of proteins than the biuret method.
reaction is strongly influenced by four amino acid residues (cysteine, cystine, tyrosine, and tryptophan) in the amino acid sequence of the protein.
Bradford protein assay was developed by Marion M. Bradford in 1976: a colorimetric protein assay, is based on an absorbance shift of the dye Coomassie Brilliant Blue G-250.
.
.
.
.
.
This video is about protein A260/280 ratio, nanodrop data interpretation, nanodrop results meaning, Nanodrop A260/280, Nanodrop protein concentration, Nanodrop dna quantification, nanodrop spectrophotometer, a260/a280 ratio rna, a260/a280 ratio higher than 2, a260/a280 nanodrop, a260/a280 value of pure rna, a260/a280 value of pure dna, Nanodrop spectrophotometry, Biomolecule measurements, DNA quantification
RNA quantification, Protein quantification, UV-Vis spectroscopy, Nucleic acid concentration, Absorbance spectroscopy, Spectrophotometric analysis
Biomolecular assays, Quantitative analysis methods, Molecular biology techniques, Laboratory instrumentation, Nanodrop spectrophotometer tutorial and Precise biomolecule quantitation.
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