You are given a pure protein sample to characterize and provided the following information: Its molar extinction coefficient, ε280, is 0.25 liters micromole-1 cm-1 in both the folded and unfolded form Its ΔGo for unfolding is 1.5 kcal/mol at 37o (where RT = 0.59 kcal/mole) A) Using a 0.5 cm pathlength cell, you measure the absorbance at 280 nm of a 20-fold dilution of your pure protein in solution (by this, we mean that 50 ul of the protein sample was diluted to a final volume of 1 ml) and find A280 = 0.40. What is the original concentration of the protein before dilution? B) What is the concentration of the unfolded form of the protein in your sample?
Proteins
We generally tend to think of proteins only from a dietary lens, as a component of what we eat. However, they are among the most important and abundant organic macromolecules in the human body, with diverse structures and functions. Every cell contains thousands and thousands of proteins, each with specific functions. Some help in the formation of cellular membrane or walls, some help the cell to move, others act as messages or signals and flow seamlessly from one cell to another, carrying information.
Protein Expression
The method by which living organisms synthesize proteins and further modify and regulate them is called protein expression. Protein expression plays a significant role in several types of research and is highly utilized in molecular biology, biochemistry, and protein research laboratories.
You are given a pure protein sample to characterize and provided the following information:
Its molar extinction coefficient, ε280, is 0.25 liters micromole-1 cm-1 in both the folded and unfolded form
Its ΔGo for unfolding is 1.5 kcal/mol at 37o (where RT = 0.59 kcal/mole)
- A) Using a 0.5 cm pathlength cell, you measure the absorbance at 280 nm of a 20-fold dilution of your pure protein in solution (by this, we mean that 50 ul of the protein sample was diluted to a final volume of 1 ml) and find A280 = 0.40. What is the original concentration of the protein before dilution?
- B) What is the concentration of the unfolded form of the protein in your sample?
1(A)
Beer-Lambert Law is often used in spectrophotometry to quantitatively analyze the concentration of a solute in a solution by measuring the absorbance of light at a specific wavelength.
The Beer-Lambert Law is expressed as follows:
Aλ = ε * c * l
Where:
- Aλ represents the absorbance of the solution at wavelength λ.
- ε is the molar absorptivity or molar extinction coefficient of the substance, which is a constant specific to the substance and the wavelength of light being used.
- c is the concentration of the substance in the solution
- l is the path length of the cuvette or container through which the light passes
Calculation of concentration of protein in cuvette:
A280 = 0.40 = (0.25 L
c= 3.2
Concentration of protein in the sample before dilution will be 20 times greater.
So, original concentration of the protein before dilution = 20*3.2
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