Methods of Protein Analysis / Determination

Protein Determination Methods:

1. Kjeldahl Method

The first step in the Kjeldahl method is the digestion of the sample in sulfuric acid with a catalyst which converts nitrogen to ammonia. The next step is the distillation of the ammonia from the previous step into a trapping solution. Lastly, the ammonia is titrated with a standard solution to determine the amount (3.1 Nitrogen Determination by Kjeldahl (rack), 2007). The amount of nitrogen is calculated using a formula. This amount is then applied to another formula to determine protein content. (Blamire, 2003).

2. Dye Binding Method

                At low pH, basic groups of protein are positively charged. These will quantitatively bind a negatively charged dye. In doing this method, you must first mix the protein, dye and buffer which has a pH of two. Then you filter or centrifuge the mixture. You then measure the optical density of the filtrate. The optical density of the dye bound protein is equal to the optical density of the dye initially and the optical density of the filtrate. The factors influencing Dye Binding Determination are temperature, non-proteins, buffer systems, and protein quality.

3. Biuret Method

                This method is a good general protein assay for batches of material for which yield is not a problem. Under alkaline conditions, substances containing two or more peptide bonds form a purple complex with copper salts in the reagent. It uses a spectrophotometer to measure the absorbance and then calculate for the amount of protein. (Caprette, 1997)

4. Lowry Method

                The principle behind the Lowry method of determining protein concentrations lies in the reactivity of the peptide nitrogens with the copper ions under alkaline conditions. It is sensitive to low concentrations of protein. A variety of compounds will interfere with the procedure like amino acid derivatives, certain buffers, drugs, lipids, sugars, salts, nucleic acids and sulphydryl reagents. (Williamson, 2000)

5. Ultra-violet Absorption (UV) at 280 nm

                Most proteins absorb ultraviolet light at a maximum of 280 nm wavelength because of the presence of Tyrosine and Tryptophan. This has been used as a rapid and fairly sensitive measure of protein concentration. However, nucleic acids absorb light at a wavelength of 280 nm, but they absorb much more strongly at 260 nm UV light. For protein it is the reverse situation. This assay uses this reverse relationship to calculate the interference of nucleic acids in the estimation of protein concentration.

                The concentration of the protein in solution is calculated according to the treatment of Warburg and Christian. With this protocol, it is not necessary to know the nucleic acid concentration of the sample to find the protein concentration of an unknown sample. This method gives considerable error with mixture containing more than 20% nucleic acids or with very turbid solutions. (determination of protein concentratoin by UV absorption)

6. Fluorescence Method

                Tyrosine and Tryptophane is a fluorescent compound. This method excites amino acids at 280 nm and measures the emission at 348 nm. It is more sensitive than UV absorption. (Protein Determinatin Methods)

7.  Combustion method

                Total protein is determined using nitrogen analysis. The sample is combusted with oxygen and the gases containing nitrogen oxides are collected in a ballast tank until a specified pressure is reached. Helium is used as a carrier and an aliquot of combustion gas containing nitrogen oxides is reduced to nitrogen. It is then passed through a tube containing magnesium perchlorate and sodium

hydroxide on a silicate carrier to remove water and carbon dioxide. The nitrogen is measured with a thermal conductivity detector using helium as a reference. Nitrogen is then converted to protein using a conversion factor. (Protein Determination by Combustion, 2009)

 Bibliography

3.1 Nitrogen Determination by Kjeldahl (rack). (2007). Retrieved September 5, 2011, from Foragetesting.org: http://www.foragetesting.org/lab_procedure/sectionB/3/part3.1.htm

Blamire, J. (2003). e-learning for Quantitative Analysis-kjeldahl method. Retrieved September 5, 2011, from science@a distance: http://www.brooklyn.cuny.edu/bc/ahp/SDKC/Chem/SD_KjeldahlMethod.html

Caprette, D. R. (1997). Biuret Protein Assay. Retrieved September 2011, from Experimental Biosciences: Introductory Laboratory - Bioc 211: http://www.ruf.rice.edu/~bioslabs/methods/protein/biuret.html

determination of protein concentratoin by UV absorption. (n.d.). Retrieved 2011, from Center for cocoa biotechnology research and development: http://www.koko.gov.my/CocoaBioTech/Protein%20Quantitation1.htm

Protein Determinatin Methods. (n.d.). Retrieved September 2011, from Ohio State University: class.fst.ohio-state.edu/fst601/food%20chemistry%20601-8.pdf

Protein Determination by Combustion. (2009). Retrieved September 2011, from United States Department of Agriculture: Food Safety and Inspection Service, Office of Public Health Science: www.fsis.usda.gov/PDF/CLG_PRO_4_03.pdf

Williamson, J. (2000). Biology 371 Independent Research week 5: Protein Determination-Lowry Procedure. Retrieved September 2011, from Davidson College: http://www.bio.davidson.edu/people/jowilliamson/Techniques/Protocolweek5.html