SPECTROPHOTOMETRIC DETERMINATION OF PROTEIN CONTENT IN THP-1 MONOCYTES/MACROPHAGES – DESCRIPTION OF THE METHOD

Jolanta Wolska, Katarzyna Janda, Izabela Gutowska

Abstract


Proteins are the basic building block of tissue, and are part of enzymes and hormones regulating many important life processes. Changes in their concentration control the metabolic processes of the cell. Quantitative determination of the protein content is divided into indirect methods (e.g. Kjeldahl method) and direct methods (buret method, Lowry, immunoenzymatic, formol method, based on incorporation of dye in the range of ultraviolet spectrophotometry, and based on the phenomenon of selective absorption of radiation in the infrared range). One of the methods for the determination of protein content is the spectrophotometric method described by Bradford. The protein concentration assay procedure utilizes the phenomenon of formation of the dye (Coomassie Brillant Blue G-250)–protein and colour intensity is proportional to the protein content in the solution. The aim of this study was to verify the usefulness of this method for determining the protein content in THP-1 cells cultured with extracts of nettle fruit stalks (Urtica dioica L.). Aqueous and alcohol extracts at two concentrations were used. It has been shown that the spectrophotometric determination of protein content by the Bradford method is an effective and accurate method for determining the concentration of protein in THP-1 macrophages. The results indicate that this method can be recommended for the determination of the protein content in other cell cultures.


Keywords


THP-1 cells; protein; spectrophotometer

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References


Murray R.K., Granner D.K., Mayes P.A., Rodwell V.W.: Biochemia Harpera. Wyd. Lek. PZWL, Warszawa 2000.

Myszka K., Kamińska B., Fraś A., Ploch M., Boros D.: Metoda Dumasa alternatywną metodą oznaczania białka w produktach roślinnych – badania porównawcze z metodą Kjeldahla. Biuletyn Instytutu Hodowli i Aklimatyzacji Roślin. 2011, 260/261, 155–161.

Sapan C.V., Lundblad R.L., Price N.C.: Colorimetric protein assay techniques. Biotechnol Appl Biochem. 1999, 29, 99–108.

Kashyap M.K., Hynd B.A., Robinson K.: A rapid and simple method for measurement of total protein in very low density lipoproteins by the Lowry assay. J Lipid Res. 1980, 2, 1, 491–495.

Enechi O.C., Nwabueze C.E.: A new colorimetric method for the determination of proteins. Adv Biol Res. 2013, 7, 5, 159–162.

Adewale I.O., Akinseye O.R., Odutayo A.I.: Protein quantitation using dyes obtained from plant materials. Adv Biosci Biotechnol. 2012, 3, 227–230.

Bradford M.M.: A Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976, 72, 248–254.

Pandey N., Budhathoki U.: Protein determination through Bradford’s method of Nepalese mushroom. Scientific World. 2007, 5, 5, 85–88.




DOI: https://doi.org/10.21164/pomjlifesci.57

Copyright (c) 2016 Jolanta Wolska, Katarzyna Janda, Izabela Gutowska

License URL: https://creativecommons.org/licenses/by-nc-nd/3.0/pl/