Measurement of concentration of protein sample using UV-Vis spectrophotometer - Biophysical Instrumentation Practical

Measurement of concentration of protein sample using UV-Vis spectrophotometer   

 

 

INTRODUCTION:

 

 

There are several ways of estimating the protein concentration such as amino acid analysis following acid hydrolysis of the protein; analyzing the changes in the spectral properties of certain dyes in the presence of proteins; and spectrophotometric estimation of the proteins in near or far UV region. Although dye-binding assays and amino acid analysis following acid hydrolysis of the protein can be used for estimating the protein concentration for both pure as well as an unknown mixture of proteins; UV spectroscopic quantitation holds good for the pure proteins. If a protein is pure, UV spectroscopic quantitation is the method of choice because it is easy and less time consuming to perform; furthermore, the protein sample can be recovered back. 

If the protein sample does not have tryptophan or tyrosine, both of which absorb at 280 nm, the concentration can still be easily measured by the Scopes Method.  In this particular method, the protein concentration is determined by the absorption at 205 nm in which the peptide bonds are analyzed directly. DNA or RNA purity can also be determined by measuring the absorption ratios of 260 to 280 nm.  This is because the nucleic acids that make up DNA and RNA absorb strongly at 260 nm.  A ratio of about 2.0 is considered "pure" for RNA while a ratio of about 1.8 is considered "pure" for DNA.  Lower ratios indicate the presence of protein. 

PRICIPLE:  

The Beer-Lambert law states that the rate at which the intensity of a monochromatic light beam decreases along the thickness of a solution that contains an absorbent of that monochromatic light is directly proportional to the concentration of the absorbing substance in the solution and is also directly proportional to the intensity of the incident monochromatic radiation. According to the Beer-Lambert equation, the amount of radiation absorbed increases with the number of absorbing molecules (molecules with the capacity to absorb light of a particular wavelength.           

                                                                      A = εbc  

                                                                     c = A/ εb 

MATERIALS REQUIRED: 

10 mg/ml BSA solution, TRIS buffer solution, Quartz Cuvettes, Micropipette, Ice bath, Spectrophotometer, protein sample.  

PROCEDURE: 

To measure the maximum wavelength of the unknown protein sample: 

1. 750microlitre TRIS buffer in two Cuvettes in UV-Vis spectrophotometer  

2. In the desktop, go to “ spectrophotometer” → “autozero” →”method” →”general” →”wavelength scan” →”instrument” → and set the start wavelength = 400nm and end  wavelength=190nm →set the baseline →”system”.   

3. Replace one of the cuvette with BSA sample.  

4. Go to “sample” →”measure”, measure the maximum wavelength.  

 

To measure the absorbance value at maximum wavelength of the unknown protein sample: 

1. In the desktop, go to “method” →”general” →”measurement” →”photometry” →”instrument” →”wavelength” →put the wavelength as the maximum observed value→ measure the absorbance value.  

OBSERVATION:  

It was discovered that the maximum wavelength was 282nm and the absorbance value at 282nm was 0.310.  

CALCULATION: 

λmax = 282nm  

A= 0.310  

ε = 43824 L mol – 1 cm – 1 

 c = A/ εb 

c = 0.310/ 43824 x 1 = 7.073 x 10-6 M = 7.073 micro Molar.  

INTERPRETATION:  

Using the UV visible spectrophotometer it can be observed that the concentration of the given protein sample is 7.073 micro molar.