Question

Introduction of Quantitative Analysis of Protein by Spectrophotometry written and not in picture format please

Answer 1

Answer :-

Because proteins absorb light at a specific wavelength, the concentration of a pure protein in solution can be directly measured using a spectrophotometer. It is important to note that direct measurement of UV at 280 nm yields highly reproducible measurements as no reagents are applied to the protein solution and during the process the protein of interest has not been changed or inactivated.

It also provides quick results, as the sample need not be incubated to complete the process.

The protein's chemical composition (i.e., the number and type of amino acids present) can affect its absorption. Since the absorption of a sample protein at 280 nm can depend on the sum of the amino acids tyrosine and tryptophan, due to their different content of tyrosine and tryptophan, it is very likely that proteins with similar molecular weight will have different absorption values.

In addition, the structure of concern of the protein may also influence the absorption of aromatic side chains by UV. As such, the ability of aromatic residues can be affected by the temperature, pH, ionic force and/or presence of detergents to absorb light at 280 nm and alter the protein's extinction coefficient.

Calculation of protein concentrations The following formula can be used to measure the concentration of a pure protein in a solution: Concentration (μg / ml)= A280 x Conversion Factor Note: Each protein has its own conversion factor. Use the A260/A280 ratio to test your protein sample for its purity.

Additionally, you can also use the following formula for calculating the protein concentrationConcentration (μg / ml)= A280/Absorbance (E280,1mg / ml) x l) Note: The absorbance coefficient varies between proteins and can be measured either by measuring the absorption of the protein in a specified concentration solution or by theoretical calculation.

The presence of nucleic acids in your protein sample can affect the precision of your measurements but the protein concentration in your sample can still be calculated regardless. C (mg / ml)= 1.55 A280— 0.76 A260 A wide range of colorimetric methods can also be used to calculate concentrations of proteins.