Lowry test

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The Lowry test is a biochemical method used to quantify proteins . The publication about the method from 1951 is the most frequently cited scientific article between 1945 and 2014 with 305,148 citations.

principle

The Lowry test is based on two reactions. The first step is based on the biuret reaction , namely the formation of a blue-violet, square-planar dye complex between the peptide bonds and copper (II) ions in an alkaline solution. In a second step, Cu (II) is reduced to Cu (I) by the peptide bond. This Cu (I) in turn reduces the yellow Folin-Ciocalteu reagent ( molybdophosphoric acid and tungstophosphoric acid / heteropoly acids ) to heteropolymolybdenum blue. The Folin-Ciocalteu reagent lowers the detection limit by a factor of 100 compared to the biuret reaction. The Lowry method is much more sensitive than the Biuret method because of its second, additional color reaction. Protein concentrations of 0.1–1 µg protein per milliliter can also be determined. However, it is more time-consuming and more prone to failure. This is disrupted by non- proteinogenic substances and the frequently used substances EDTA , Triton X-100 or ammonium sulfate .

The Lowry reagent consists of the aqueous stock solutions A (100 g / L sodium carbonate , 0.5 M sodium hydroxide ), B (10 g / L copper sulfate pentahydrate) and C (20 g / L potassium tartrate ), which are mixed in a ratio of 20 : 1: 1. The Lowry reagent is mixed 1: 1 with the sample and incubated for 15 minutes at room temperature. The dye is then formed by adding the Folin-Ciocalteu reagent diluted 1:10 in a ratio of 2: 3 and incubating at room temperature for 45 minutes. The resulting intense color is measured for quantitative determination of the protein concentration by means of photometry at 750 nm , 650 nm or 540 nm.

Some improvements and further developments have been described in the literature. In 1972 Hartree presented a variant of the Lowry method which, among other things, addresses the high susceptibility to failure. The proportion of the amino acid cysteine contributes to the blue color. With regard to the time development of the blue coloration after the addition of the Folin-Ciocalteu reagent, Lowry recommended a waiting time of 30 minutes and then measuring the extinction at 750 nanometers . The intensity of the blue color increases between 30 and 120 minutes; it remains stable between 120 and 240 minutes and then decreases again. In addition, it is advisable to prepare the Lowry reagents with water instead of sodium hydroxide , but the protein solutions in 1 M sodium hydroxide. Furthermore, a wavelength of 660 nanometers is more suitable than the usual 750 nanometers for measuring the extinction by photometry.

As an alternative, there is the simpler Bradford protein determination , which is similarly sensitive. The advantage of the Lowry method over the Bradford method is that it can also be used to determine protein concentrations in solutions that contain sodium lauryl sulfate (SDS), which would hinder the Bradford determination. Alternatively, the Cu (I) can be converted into a violet complex using bicinchonic acid (BCA) in the BCA test , which is determined photometrically - another modification of the Lowry test.

Individual evidence

  1. ↑ Oliver H. Lowry et al. : Protein measurement with the Folin phenol reagent. In: J. Biol. Chem. Volume 193, No. 1, 1951, pp. 265-275. PMID 14907713 PDF .
  2. ^ Richard Van Noorden, Brendan Maher, Regina Nuzzo: The top 100 papers. In: Nature. 514, 2014, pp. 550-553, doi : 10.1038 / 514550a .
  3. ^ N. Kresge, RD Simoni, RL Hill: The Most Highly Cited Paper in Publishing History: Protein Determination by Oliver H. Lowry . In: Journal of Biological Chemistry . 280, No. 28, 2005, p. E25.
  4. ↑ Hubert Rehm : Simple and yet so complicated. In: Laborjournal . Issue 7–8, 2008, p. 38.
  5. ^ AG Gornall, SJ Bardawill, MM David: Determination of serum proteins by means of the biuret reaction. In: J Biol Chem. Volume 177 (2), 1949, pp. 751-766. PMID 18110453 .
  6. ↑ Alexander J. Ninfa: Fundamental Laboratory Approaches for Biochemistry and Biotechnology . John Wiley & Sons, INC., United States of America 2010, ISBN 978-0-470-08766-4 , p. 112.
  7. ^ A b c d Alfred Pingoud : Working methods of biochemistry. Walter de Gruyter, 1997, ISBN 978-3-110-16513-5 , p. 152.
  8. ↑ a b c F. Lottspeich, JW Engels, A. Simeon (Ed.): Bioanalytik. 2nd Edition. Spectrum Academic Publishing House, 2006, ISBN 3-8274-1520-9 . P. 38.
  9. ^ EF Hartree: Determination of protein. A modification of the Lowry method that gives a linear photometric response. In: Anal. Biochem. Volume 48, 1972, pp. 422-427. PMID 4115981 doi : 10.1016 / 0003-2697 (72) 90094-2 .
  10. ↑ JD Everette, QM Bryant, AM Green, YA Abbey, GW Wangila, RB Walker: Thorough study of reactivity of various compound classes toward the Folin-Ciocalteu reagent. In: Journal of agricultural and food chemistry. Volume 58, number 14, July 2010, pp. 8139-8144, doi : 10.1021 / jf1005935 , PMID 20583841 , PMC 4075968 (free full text).
  11. ^ Christopher Pomory: Color development time of the Lowry protein assay. In: Anal. Biochem. Volume 378, No. 2, 2008, pp. 216-217. PMID 18448065 doi : 10.1016 / year from 2008.04.015 .
  12. ↑ JR Dulley and PA Grieve: A simple technique for eliminating interference by detergents in the Lowry method of protein determination. In: Anal Biochem. Volume 64, No. 1, 1975, pp. 136-141. PMID 1137083 doi : 10.1016 / 0003-2697 (75) 90415-7 .
  13. ↑ PK Smith et al. : Measurement of protein using bicinchoninic acid. In: Anal. Biochem. Volume 150, No. 1, 1985, pp. 76-85. PMID 3843705 doi : 10.1016 / 0003-2697 (85) 90442-7 .