Protein Quantification by UV-Visible Spectrophtometer

Protein quantification by spectrophotometer is measurement of total pure protein in a sample on the basis of Beer Lambert Law. This article explains the structure , principle ,procedure and protocol of protein quantification by Spectrophotometer.

Working of spectrophotometer

Spectrophotometer consists of two main devices i.e. A spectrometer (produces and disperse the light) and photometer (detects light intensity) hence the name spectrophotometer Figure 2.

Following are the functions of the each part of the spectrophotometer (Figure 3):

To understand the principle of Spectrophotometer you must know Beer Lambert Law.

Beer-Lambert Law

If light is passed through a solution of a biomolecule, it will absorb a fraction of light ONLY at a specific wavelength. E.g. Tryptophan (a non-polar aromatic amino acid) absorbs maximum light at 280nm, Phenol at 270nm and DNA at 260nm. This wavelength at which the absorbance is maximum is called Lambda max (λ_max) of that particular molecule. The ratio of Intensity of incident light (I_o ) to the intensity of transmitted light (I) is called transmittance i.e. T=I/I_o as shown in Fig. 01.

 Log of Inverse of transmittance is called absorbance (A) or optical depth or misleadingly optical density (OD). Hence,

A= log I_o / I

This was all about absorption of light by a bio-molecule at a specific wavelength but the amount of the light absorbed by the solution of a bio-molecule depends upon

1. The thickness of the absorbing layer of the solution (Path Length, l)
2. Concentration of absorbing molecule (c)

The relationship between path length and concentration of a bio-molecule is called Beer Lambert Law which states “that absorbance of a molecule is directly proportional to its concentration and path length”. It can be written as :

A= EcL

A = Absorbance (dimensionless)
c = Concentration (mol/L)
l = Path length (cm)
E = Molar Absorptivity or Molar extinction coefficient (Lmol^-1cm^-1)

A is monotonically increasing function the path length, if the path length is zero, absorbance is zero.  E is dependent upon the nature of the molecule, solvent and wavelength. Hence, E has a specific value for a specific molecule. E.g. tryptophan has E= 5690 L·mol^-1·cm^-1

Assumptions of Beer Lambert law.

1. The incident light is parallel (Travelling a perfect straight line through the solution) as well as monochromatic (of a single wavelength)
2. Solute and solvent are randomly oriented (Longer path length will give you higher absorbance)

Principle of UV-Visible Spectrophotometer

Aromatic amino acids (tryptophan, tyrosine, Phenylalanine ) and cysteine, disulfide bonded cysteine residues present in a protein have bonding and non-bonding electrons which can absorb energy in the form of ultraviolet or visible light to excite these electrons to higher anti-bonding molecular orbitals.There are four possible types of transitions (π–π*, n–π*, σ–σ*, and n–σ*), and they can be arranged on the basis of energy absorbed as follows: σ–σ* > n–σ* > π–π* > n–π*. When the light passes through the sample it is absorbed and some of the light is transmitted to the detector. The spectrophotometer compares the incident light and transmitted light and gives absorbance. Following is the table showing the excitation wavelength and emission wavelengths of aromatic amino acids.

Protocol

In practical, the path length is kept constant, mostly 1cm which is actually the width of the cuvette. Hence, the absorbance becomes merely dependent on the concentration of the solution. Concentrated solution will give higher absorbance, dilute solution will give lower value of Absorbance. On the basis of this fact, a standard Curve can be plotted. A standard curve is a graph between the absorbance on y-axis and known concentration of a biomolecule (e.g. proteins) on x-axis. Then, the absorbance of the sample of unknown concentration is taken by spectrophotometer; this value is compared with the standard curve to find the concentration of the unknown sample

Protein sample can be quantified by two approaches:

By Beer Lambert Law

• Measure the Absorbance (A) of the sample by spectrophotometer at 280nm.
• Find its Molar extinction coefficient from literature.
• Path Length (l) is 1cm by default.
• You can use the equation A = Ecl to find the c.

By Standard Curve

Protein concentration can be found by comparison with a calibration curve prepared from measurements with standard protein solutions.

1. For calibrating with standards, use the 3 mg/ml standard protein solution to prepare dilutions of 20, 50, 100, 250, 500, 1000, 2000, and 3000 µg/ml in the same solvent as used to prepare the sample protein (Positive Control) as shown in following table.
2.  Prepare a blank consisting of solvent alone (Negative Control).
3. Turn on the spectrophotometer and let it warm up for 30min.
4. Take 1ml blank and place it in spectrophotometer, zero the spectrophotometer with solvent blank.
5. Take absorbance at 280nm of the prepared standard protein as above table.
6. Make standard curve.
7. Calculate the Absorbance of the unknown sample, correspond it on the graph to know its concentration as shown in the following graph.

H2O	Standard Protein	Final Volume	Dilution Factor
990ul	10ul	1000ul (1ml)	100
980ul	20ul	1000ul (1ml)	50
970ul	30ul	1000ul (1ml)	33.33
960ul	40ul	1000ul (1ml)	25
950ul	50ul	1000ul (1ml)	20

The concentration that you get from this will be in mg/ml units. You can change it into mole by the following formula:

FAQs

How to choose the blank?

Blank solution is the combination of all the substances/solutions/buffer except your sample (protein) which you want to quantify.

What are the applications of Spectrophotometry?

By using different approaches of using spectrophotometer you can do the following experiments:

What is protein quantification?

Protein quantification or total protein content by colorimetric methods such as absorbance at 280, Bradford or BCA assays and quantitative amino acid analysis. Protein quantification is necessary to understand the total protein content in a sample or in a formulated product.

The standard curve is not a straight line. What should I do?

There must be an error in handling or with spectrophotometer or while making standard protein solution. You can join the maximum points to get a straight line and omit the point which are out of the trend.

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