Question

What do the terms transmittance and absorbance mean in terms of sample concentration quantification?

The BCA assay is a common assay for protein quantification. Explain how it works for this purpose.

Answer 1

Beer Lambert's law

Absorption of light when it is passed through a sample is directly proportional to the concentration, path length and the molar absorbtivity of a sample.

A = bc

A= Absorbance, = molar absorbtivity L mol^-1 cm^-1, c = concentration mol L^-1.

# Amount of light passed through the sample is called as transmittance. If no light is absorbed then 100% transmittance will be there.

T= I / I_0, T= transmittance, I =intensity of transmitted light, I₀ = intensity incident light.

# Amount of light absorbed by the sample is called as absorbance. If all the light is absorbed then the transmittance will be zero.

A = log₁₀ I₀ / I

So absorbance increases with the concentration of the solution.

BCA (BiCinchoninic Acid) assay principle.

This assay is based on two steps of reaction.

# The peptide bonds in the protein reduce Cu²⁺ ions from the copper(II) sulphate to Cu⁺. The amount of Cu²⁺ reduced is proportional to the amount of protein present in the sample.

# Two molecules of bicinchoninic acid chelate with one molecule of Cu⁺ ion, forming a purple coloured complex that strongly absorbs light at 562 nm.

So the amount of colour change is proportional to concentration of protein.