Lab 2 Questions

Infrared region (IR) wavelengths

0.700-500 μm

If a sample has A280= 0.5 and A260= 0.04, what would be the protein concentration (mg/ml)?

0.72

Radio wave region Wavelength:

1 - 5 m

Two tubes, tube 1 containing 0.1 ml of a unknown protein sample and tube 2 containing 0.1 ml of 2 mg/ml BSA standard were subjected for protein concentration by the Biuret method. Absorbance were 0.25 and 0.2 for tube 1 and tube 2, respectively. What is the concentration of protein in the unknown sample in mg/ml?

2.5 mg/mL

UV region Wavelength:

200-400 nm

Visible region Wavelength:

400-800 nm

A solution of protein 'X' in a 1 cm cuvette has A280 = 0.41, what is the concentration of protein solution? (Molar extinction coefficient of protein X= 8.2E3

50 μM

A solution of Riboflavin 0.5A has in a 1.0 cm cuvette. Calculate the concentration of Riboflavin in the solution. E=1.22x104

Ans: c = A/El given, A = 0.5; E = By substituting these values in the above formula: 4.1x10^-5

The absorbance of a 5 x 10-4 M solution of Tyrosine, at a wavelength of 280 nm is 0.75. The light path length of the cuvette is 1 cm. Calculate the molar absorption coefficient, E.

E = A/lc or 0.75/ (1 cm)(5 X10-4 mole/L) = 1500 L/mole/cm

Biuret method:

The Biuret method of protein determination is based on the interaction of Cu2+ with protein. This reaction is dependent in part on peptide bonds and not solely on amino acid moieties. The reaction is simple and gives a linear correlation between absorbance and protein concentration over a wide range. However, it is not as sensitive as the Bradford and Lowry methods of protein determination.

Bradford method:

This is a rapid and reliable dye-based assay for protein content. It is based on binding of Coomassie Blue dye to the protein. This is an extremely sensitive assay which detects as little as 1 μg/ml of protein. This method is not only rapid but has very few interference problems arising from non-protein components in the assay mixture. The binding of Coomassie Blue dye to protein in acidic solution causes a shift in wavelength of maximum absorption (λmax) of dye from 465 to 595 nm. The absorption at 595 nm is directly related to the concentration of protein.

Lowry method

This is a standard method for quantitating protein content. Two color reactions take place: a) Alkaline Cu2+ reacts with peptide bonds in proteins (Biuret reaction), and b) the reaction of complex salt of phosphomolybdotungstate (phenol reagent), which gives an intense blue-green color with Biuret complexes of tyrosine and tryptophan. -Biochemical assay for determining the total level of protein in a solution. The total protein concentration is exhibited by a color change of the sample solution in proportion to protein concentration, which can then be measured using colorimetric techniques.

According to the Beer-Lambert Law, what are the factors that affect the absorbance?

both length of medium and concentratio

For UV region absorption, which light source will be used?

deuterium lamp

Which type of cuvette would you use to measure absorbance at 260 nm?

quartz cuvette

Which of the following statement is related to the principle of Bradford method of protein determination?

shift in a wavelength of maximum absorption of dye from 465 to 595 nm