Modified Lowry Protein assay
Michał Kaczmarek
Keywords: Lowry, protein concentration,
equipment required:
 spectrophotometer capable of measuring
absorbance in the 650 nm region.
 disposable cuvettes for spectrophotometer
required reagents:
 Reagent A – consist of 2% solution of Na2CO3 in 0,1 mol/dm3 NaOH
 Regent B – consist of 0,5% solution of CuSO4 x 5H2O in 1% sodium citrate CH3COONa
 Reagent C – to prepare reagent C mix both reagents (A and B) in appropriate molar ratio
5:1 (v/v)
 protein standards BSA (bovine serum albumin) solutions at the concentration ranging from
0 µg/ml to 50 µg/ml
 Folin-Ciocalteau reagent
execution time: 60 min.
Procedure Description:
The Lowry assay (1951) is an often-cited general use protein assay. Under alkaline conditions
the divalent copper ion forms a complex with peptide bonds in which it is reduced to a monovalent
ion. Monovalent copper ion and the radical groups of tyrosine, tryptophan, and cysteine react with
Folin reagent to produce an unstable product that becomes reduced to molybdenum/tungsten blue.
Procedure:
1. Gently mix the Folin-Ciocalteau reagentin the.
2. Prepare protein standards in appropriate buffer (0,1 M or 0,2 M phosphate buffer
pH 7,2) ranging from 0 µg/ml to 50 µg/ml using a BSA standards.
3. Add 0,4 ml of each protein standard to separate tubes. To the tubes used as the blanks,
add 0,4 ml of buffer.
4. Prepare the unknown samples in an appropriate dilution.
5. To each tubes, add 2 ml of the Reagent C and mix.
6. Let the samples incubate at room temperature for 10 minutes.
7. To each tubes, add 0,2 ml of the Folin-Ciocalteau reagent and mix
8. Incubate samples in 37°C for 30 minutes.
9. Transfer samples into cuvettes
10. Measure the absorbance at 650 nm.