Protein Measurement by UV/Visible spectroscopy

Advanced Biotechnology Lab I Florida Atlantic University January 14, 2008

0 14.The purification table revisited Step Total activity (U) 3000 2400 1440 1000 Total protein (mg) 15000 4000 500 125 Specific activity* (U/mg) 0.9 8.6 2.2 0. an accurate assay for total protein is necessary .0 Yield† (%) 100 80 48 33 Purification factor‡ 1 3.5 40 1 2 3 4 *Specific activity = Total activity / Total protein † Yield = Total activity at step “x” divided by total activity at step 1 ‡ Purification factor = Specific activity at step “x” divided by specific activity at step 1 In order to track purification of your protein.

tryptophan. Cu2+ forms a complex with peptide nitrogens. Aromatic residues of proteins absorb light near 280 nm. lysine. primarily arginine but also histidine.Common protein assays Assay Biuret Mechanism Under alkaline conditions. Lowry Bradford UV . Absorbance is read at 595 nm. which in turn absorbs light at 550 nm As with Biuret except that an additional reagent (Folin) is used to increase sensitivity Under acidic conditions. and phenylalanine residues react with Coomassie blue dye. tyrosine.

4 7. The Bradford assay is particularly sensitive to bovine serum albumin (BSA). These assays are only 100% accurate when calibrated against standards of the same protein.3 4.2 12.6 7.4 11.4 16.3 10.1 5.8 8.5 8.3 9.9 15.7 10.8 21.7 Lowry mg/ml 5.1 7.2 9.7 9.9 15.3 All proteins solutions in the table are 10 mg/ml.4 15.8 11.8 9.0 19.7 25. You must take this into account when using BSA as a standard.3 9.9 12.8 11. Differing assay numbers result from varied amino acid composition between proteins.3 11.1 25.9 9.2 6.4 13.2 9.4 7.0 8.7 4. .Colorimetric assays are affected by amino acid composition Protein Alcohol dehydrogenase Bovine serum albumin Cytochrome c Ovalbumin Fibrinogen Gamma globulin (rabbit) Hemoglobin (bovine) Histones Lysozyme Myoglobin Pepsin Ribonuclease Trypsin Thyroglobulin Biuret mg/ml 5.8 9.7 10.8 8.9 20.2 Bradford mg/ml 7.

Bradford assay • Coomassie blue has an absorbance peak at 465 nm • Once bound to protein. the absorbance peak is at 610 nm • The greatest difference in absorbances is found at 595 nm .

. depending on the percentage of amino acids which react with the Coomassie dye.Bradford assay (cont.) Standard curves vary from one protein to another.

300 5.400 8.000 1.Aromatic amino acids absorb strongly near 280 nm Residue Tryptophan λmax 280 219 274 222 257 206 211 250 ε 5.900 300 Tyrosine Phenylalanine Histidine Cysteine Beer-Lambert Law: Absortivity scale in figure is logarithmic εTrp>>εTyr>>εPhe A=ε cl .000 200 9.600 47.

as in crude cell lysates. .UV absorbance of DNA and protein The spectra of DNA and protein overlap. so this must be taken into consideration when samples contain both.

Protein assay by UV absorbance The relationship between protein concentration and UV absorbance is complicated by a number of factors:  Different amino acids absorb at different wavelengths  The extinction coefficients differ widely  The amino acid composition of proteins varies widely  Nucleic acids absorb strongly near 260 nm However. l is path length in cm (usually 1) . 55 A280−0 .76 A260  l When there are no contaminating nucleic acids When nucleic acids are present c is concentration in mg/ml. two approximation formulas have been derived empirically: c= A280 l c=  1.

 DNA can be “salted out” by ammonium sulfate precipitation or spun down by high-speed centrifugation. then there is likely to be a large amount of DNA in your sample.  The Biuret and Lowry methods are not as fast as the Bradford assay and hence are not as widely used.Which assay to use?  If you are analyzing crude cell lysates or tissue homogenates that have not been centrifuged at high speed (100. The Bradford assay is best in this situation as the reagent only reacts with protein. . and the UV protein assay is then suitable.000 X g).

Sign up to vote on this title
UsefulNot useful