Lowry protein assay

The protein determination by the Lowry method is a method to quantitatively determine proteins.

It is based on two reactions:

• The first step is based on the biuret reaction, namely the formation of a blue-violet, square-planar complex between the peptide bonds and the copper (II ) ions in alkaline solution.
• In a second step, the Cu ( II) to Cu (I ) is reduced. This Cu (I) in turn reduces the yellow Folin -Ciocalteu reagent ( molybdophosphoric acid and tungstophosphoric acid / heteropolyacids ) to molybdenum blue. The resultant intense blue color is measured to quantify the protein concentration by photometry at 750 nm, 650 nm or 540 nm.

The Lowry method is much more sensitive than the Biuret method for their second additional color reaction. It can also protein concentrations of 0.1-1 micrograms of protein per milliliter are determined 1-10. However it is time-consuming and prone to failures. These interfering substances and non- proteinogenic EDTA, Triton X-100 and ammonium sulfate.

In the literature, some improvements and enhancements have been described. So Hartree in 1972 presented a variant of the Lowry method that addresses, among other things, the high susceptibility. With respect to the time development of the blue color after the addition of Folin- Ciocalteu reagent recommended Lowry a waiting time of 30 minutes and then the absorbance at 750 nm measured. A recent study by Christopher Pomory shows, however, that the intensity of the blue coloration is still increasing in the period between 30 and 120 minutes remains stable 120-240 minutes and then decreases again. In addition, recommend it to the Lowry reagents with water instead of sodium hydroxide should be recognized, however, the protein solutions in 1 M sodium hydroxide. Furthermore, were used to measure the absorbance of 660 nm suitable than the usual 750 nanometers.

An alternative is the simpler protein determination in Bradford, which is a similar sensitivity. The advantage of the Lowry method compared to the method of Bradford is that even protein concentrations in solution can be determined by their containing sodium lauryl sulfate (SLS ) included, which would hamper the determination according to Bradford. Alternatively you can also the Cu ( I) by means of bicinchoninic acid (BCA ) to convert to an intensely - pink complex which is determined photometrically.

Trivia

Due to the widespread use of this method is to 2005 at the listed publication of Oliver Lowry most often cited for the general.