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Friday, April 29, 2011

Principle of Protein Assays


Principle of Protein Assays - Fc -Lowry -Bradford assays

Principle of Protein Assay

Numerous spectrophotometric methods have been developed to estimate the amount of protein in a sample. Proteins are chromophores with absorption maximum in the UV range.Some proteins, such as cytochromes and hemoglobin, will have distinct spectral characteristics due to prosthetic groups. In addition, several indirect ways to measure protein concentrations
spectrophotometrically have been developed.

UV Absorption

 A simple method to measure protein concentration is to determine the absorption at 280nm. Tyrosine and tryptophan residues which have Amax at 275 and 280, respectively, are responsible for this absorption. The distribution  of tyrosine and tryptophan is fairly constant among proteins so it is not absolutely necessary to determine an extinction coefficient for each individual protein. Typically, a 1 mg/ml protein solution will result in an A280 of approximately one (1). In the case of purified proteins the exact extinction coefficient will depend on the exact amount of tyrosine and tryptophan in that particular protein. For example, a 1 mg/ml IgG solution has an A280 of approximately 1.5. The simplicity and ability to completely recover the sample are the major advantages of this method. The lower limit of sensitivity for UV absorption is 5-10 µg/ml. 

 A potential problem with using A280 values to calculate protein concentration is the absorption due to contaminating substances, and in particular, nucleic acids which have an Amax at 260 nm. It is possible to use correction factors that permit the determination of protein concentrations. A particularly convenient formula is:  

   (A235 - A280)/2.51 = mg/ml protein.

Indirect spectrophometric assays (eg., Lowry, Bradford) for the determination of protein concentrations overcome some of the problems associated with interfering substances in protein samples. However, the measured protein cannot  be recovered in such assays and they take longer to perform. 

Folin-Ciocalteu or Lowry

 Historically, one of the most widely used protein assays was the Lowry assay. This assay is a modification of a previous assay known as the Biuret. In the Lowry assay proteins react with alkaline Cu2+ reducing it to Cu+. The reduced Cu+ and the side-chain (R) groups of tryptophan, tyrosine and cysteine react with the Folin-Ciocalteu reagent (complex of inorganic salts) to form a blue color that is proportional to the amount of protein. The A600-750 is determined and protein concentration is calculated from a standard curve. The assay is linear 1-300  µg and the lower limit of sensitivity is 1-5  µg/ml. Substances in the sample such as detergents can interfere with the results and therefore appropriate controls and blanks need to be carried out.

Bradford or Coomassie Blue G-250


Protein assay

The Bradford assay has replaced the Lowry as the standard protein assay. The major advantage is that it is carried out in a single  step and that there are very few interfering substances. The principle of the assay is based on a shift of the Amax of the Coomassie-blue (G-250) dye from 465 nm to 595 nm in the presence of protein due to a stabilization of the anionic form of the dye. The dye reacts primarily with arginine residues and to lesser extent with his, lysine, tyrosine, tryptophan and phenylalanine.  Protein concentrations are determined by developing a standard curve with known amounts of proteins and extrapolating the absorbance
values of the samples. The standard curves are not linear over a wide range of protein concentrations. This assay can also carried out in 96-well plates and read on automated ELISA readers. Programs are available that will automatically calculate the protein concentrations
based upon a standard curve.

Assay Of Specific Proteins

In addition to measuring the total amount of protein, it is often necessary to estimate the amount of a specific protein in a mixture of proteins. Measuring a specific protein will depend upon the availability of an assay that is specific for the protein of interest. Protein assays should be practical in addition  to being specific and accurate . Typically protein assays are based upon the biological activity of the protein of interest. For example, enzyme assays will detect the conversion of a substrate to a product. Enzymes assays can be based upon colorimetric, fluorescent or radioactive substrates (or products). Many proteins bind to ligands or other substances and this binding activity is measured. Bioassays measure a change in some biological property (eg., stimulation of cell division). In cases where the protein of interest has no measurable activity or the activity is unknown it may be possible to generate antibodies against the protein and develop an immunoassay (to be discussed later). If antibodies against such a  protein are not available, the assay may simply be the amount of a protein band on a Commassie blue-stained gel following electrophoresis.

Reference

Lecture Notes for Methods in Cell Biology