Estimation of Protein Concentration by Spectrophotometry and Gel Filtration Chromatography
Full Report on
Exercise 4.2
ESTIMATION OF PROTEIN CONCENTRATION BY SPECTROPHOTOMETRY
And
Exercise 4.3
GEL FILTRATION CHROMATOGRAPHY
Joel Don M. Untalan
CHEM 160.1 – 1L
AY 2013 – 2014
Groupmates:
Sonette Yao
Kristopher Quilan
Laboratory Instructor
Carmelo C. Briones
I. Introduction Analyzing proteins in determination of protein concentration by spectrophotometry is important. It determines to what concentration of a certain protein is in a crude sample. In this technique, a wide range of biomolecules, particularly proteins, absorb light at characteristic wavelengths. Measurement of light absorption by a spectrophotometer is used to detect and identify molecules and to measure their concentration in solution at a given wavelength is related to the thickness of the absorbing layer (path length) and the concentration of the absorbing species. The two relationships are combined to form Lambert-Beer Law, which is A= abc wherein A is for absorbance, a is the proportionality constant, b is the length of light path through the solution, and c is the concentration of the absorbing species in the solution. The Lambert-Beer law assumes that the incident light is parallel and monochromatic meaning a single wavelength, and that the solvent and solute molecules are randomly oriented. Based also from Lambert-Beer law, absorbance, A, is directly proportional to the concentration of the absorbing solute.
Size exclusion chromatography, or famously known as gel filtration chromatography is used in separating proteins by its molecular weight or size. This technique can determine the molecular weight of the protein of interest in crude sample. In this experiment, the students will determine the absorbance of each protein inside the crude albumin sample and determine the molecular weight of albumin obtained.
The objectives of these exercises are:
To be able to know the protein concentration of the albumin.
To be able to know the relationship of
Exercise 4.2
ESTIMATION OF PROTEIN CONCENTRATION BY SPECTROPHOTOMETRY
And
Exercise 4.3
GEL FILTRATION CHROMATOGRAPHY
Joel Don M. Untalan
CHEM 160.1 – 1L
AY 2013 – 2014
Groupmates:
Sonette Yao
Kristopher Quilan
Laboratory Instructor
Carmelo C. Briones
I. Introduction Analyzing proteins in determination of protein concentration by spectrophotometry is important. It determines to what concentration of a certain protein is in a crude sample. In this technique, a wide range of biomolecules, particularly proteins, absorb light at characteristic wavelengths. Measurement of light absorption by a spectrophotometer is used to detect and identify molecules and to measure their concentration in solution at a given wavelength is related to the thickness of the absorbing layer (path length) and the concentration of the absorbing species. The two relationships are combined to form Lambert-Beer Law, which is A= abc wherein A is for absorbance, a is the proportionality constant, b is the length of light path through the solution, and c is the concentration of the absorbing species in the solution. The Lambert-Beer law assumes that the incident light is parallel and monochromatic meaning a single wavelength, and that the solvent and solute molecules are randomly oriented. Based also from Lambert-Beer law, absorbance, A, is directly proportional to the concentration of the absorbing solute.
Size exclusion chromatography, or famously known as gel filtration chromatography is used in separating proteins by its molecular weight or size. This technique can determine the molecular weight of the protein of interest in crude sample. In this experiment, the students will determine the absorbance of each protein inside the crude albumin sample and determine the molecular weight of albumin obtained.
The objectives of these exercises are:
To be able to know the protein concentration of the albumin.
To be able to know the relationship of
References: Sabularse, V. C, et al. (2011). Laboratory Instruction Manual for CHEMISTRY 160.1 Introductory Biochemistry. Biochemistry and Agricultural Chemistry Division. Institute of Chemistry. CAS. UPLB. pp. 17-26. Cox, M.M., & Nelson, D.L. (2005). Lehnninger Principles of Biochemistry (4th ed.).