The Spectrophotometer Determination of Protein Concentrations and the Effects Sodium Dodecyl and Triton X100 Have on Protein Concentration.
The Spectrophotometer Determination Of Protein Concentrations And The Effects Sodium Dodecyl Sulphate And Triton X-100 Have On Protein Concentration.
INTRODUCTION
Spectroscopy is used as a collective term for all the analytical techniques based on the interaction of light and matter. Spectrophotometry is one of the branches of spectroscopy where we measure the absorption of light by molecules that are in a gas or vapour state or dissolved molecules/ions (Tombs, et.al, 1959). Spectroscopy is the use of electromagnetic radiation by a sample in order to identify components of the sample (qualitative analysis) or to measure the amount of material in that sample (quantitative analysis) (Tombs, et.al, 1959). Absorption is a process in which matter captures electromagnetic radiation and converts the energy of the photon to internal energy (Pavia et.al, 2009). Molecules strongly absorb light only in some regions of the electromagnetic spectrum (Tombs et. al, 1959). The spectrum ranges from short wavelengths (x ray and gamma rays) to long wavelengths (microwaves and broadcast radio waves). The electromagnetic wavelength range is between 400nm – 800nm (Pavia et.al, 2009). A photon carries a specific amount of energy defined by it wavelength. Molecules only absorb a photon if the energy it carries matches a certain amount the molecule can use (Clark et.al, 1993). In the Ultraviolet – Visible light (UV-VIS) region of the spectrum this energy corresponds to electronic excitations, which are the promotion of an electron from an occupied orbital to an unoccupied orbital (Pavia et.al 2009).
In this investigation, the aim is to determine unknown protein concentrations from the Bovine Serum Albumin (BSA) standard curve and observe how the protein concentration of BSA and Lysozyme is affected by the addition of detergents, Sodium Dodecyl Sulphate (SDS) and Triton X-100. This study involves two procedures. Firstly a protein assay needs to be carried out in order to investigate the
INTRODUCTION
Spectroscopy is used as a collective term for all the analytical techniques based on the interaction of light and matter. Spectrophotometry is one of the branches of spectroscopy where we measure the absorption of light by molecules that are in a gas or vapour state or dissolved molecules/ions (Tombs, et.al, 1959). Spectroscopy is the use of electromagnetic radiation by a sample in order to identify components of the sample (qualitative analysis) or to measure the amount of material in that sample (quantitative analysis) (Tombs, et.al, 1959). Absorption is a process in which matter captures electromagnetic radiation and converts the energy of the photon to internal energy (Pavia et.al, 2009). Molecules strongly absorb light only in some regions of the electromagnetic spectrum (Tombs et. al, 1959). The spectrum ranges from short wavelengths (x ray and gamma rays) to long wavelengths (microwaves and broadcast radio waves). The electromagnetic wavelength range is between 400nm – 800nm (Pavia et.al, 2009). A photon carries a specific amount of energy defined by it wavelength. Molecules only absorb a photon if the energy it carries matches a certain amount the molecule can use (Clark et.al, 1993). In the Ultraviolet – Visible light (UV-VIS) region of the spectrum this energy corresponds to electronic excitations, which are the promotion of an electron from an occupied orbital to an unoccupied orbital (Pavia et.al 2009).
In this investigation, the aim is to determine unknown protein concentrations from the Bovine Serum Albumin (BSA) standard curve and observe how the protein concentration of BSA and Lysozyme is affected by the addition of detergents, Sodium Dodecyl Sulphate (SDS) and Triton X-100. This study involves two procedures. Firstly a protein assay needs to be carried out in order to investigate the
References: Clark, B.J., Frost, T. and Russell, M.A. (1993), UV Spectroscopy: techniques, Instrumentation, Data Handling, Springer, Great Britain, pp17-35. Nelson, C.A., (1971), The Binding of detergents to Proteins, Journal of Biological Chemistry, Vol 246, pp3895-3901. Pavia, D.L., Lampman, G.M. and Kriz, G.S., (2009), Introduction to Spectroscopy, 4th Edition, Cengage Learning, pp87-104. Rachele, R., Loo, O., Dales, N. And Andrews, P.C., (1994), Effects of Detergents on Proteins Analyzed by Electrospray Ionization. Methods in Molecular Biology, Vol 61, pp141-160. Tombs, M.P., Souter, F. And Maclagan, N.F. (1959). Spectrophotometric Determination of Protein at 210mμ, J.biol.Chem. Vol 73, pp 167-171. Williams, D.H and Fleming, I. (1980), Spectroscopy Methods in Organic Chemistry, 3rd Edition, Mc Graw-Hill, London. Voet, D. Voet, J.G and Praat, C.W. (2008) Principles of Biochemistry 3rd Edition John Wiley and Sons Ltd, New Jersey. pp 102-103.