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ýBradford – Protein Determinationý The Protein Purification Facility
The Wolfson Centre for Applied Structural Biology
The Hebrew University of Jerusalem
Dr. Mario Lebendiker
mariol@mail.ls.huji.ac.il  Tel: 972-2-6586920 

Lowry – Protein Determination
(From Protein Protocols on CD-ROM Humana Press, 1998 - Section 1-2 The Lowry Method for Protein Quantitation Jakob H. Waterborg and Harry R. Matthews)
(Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275)

Introduction

The sensitivity of the procedure of Lowry is moderately constant from protein to protein, and it has been so widely used that Lowry protein estimations are a completely acceptable alternative to a rigorous absolute determination in almost all circumstances where protein mixtures or crude extracts are involved.

The method is based on both the Biuret reaction, where the peptide bonds of proteins react with copper under alkaline conditions producing Cu+, which reacts with the Folin reagent, and the Folin-Ciocalteau reaction, which is poorly understood but in essence phosphomolybdotungstate is reduced to heteropolymolybdenum blue by the copper-catalyzed oxidation of aromatic amino acids. The reactions result in a strong blue color, which depends partly on the tyrosine and tryptophan content. The method is sensitive down to about 0.01 mg of protein/mL, and is best used on solutions with concentrations in the range 0.01-1.0 mg/mL of protein.

Materials

1. Complex-forming reagent: Prepare immediately before use by mixing the following three stock solutions A, B, and C in the proportion 100:1:1 (v:v:v), respectively.
        Solution A: 2% (w/v) Na2CO3 in distilled water.
        Solution B: 1% (w/v) CuSO4·5H2O in distilled water.
        Solution C: 2% (w/v) sodium potassium tartrate in distilled water.
2. 2N NaOH.
3. Folin reagent (commercially available). Dilute 1:2 in H2O before use.
4. Standards: Use a stock solution of standard protein (e.g., bovine serum albumin fraction V) containing 4 mg/mL protein in distilled water stored frozen at -20°C. Prepare standards by diluting the stock solution with distilled water as follows:

        stock solut.,ul             0   1.25   2.5   6.25   12.5    25     62.5     125   250
        water, ul                  500   499   498   494    488    475    438     375   250
        Prot.conc., ug/ml       0      10     20     50    100     200    500   1000 2000

Method

1. To 0.2 mL of sample or standard add 1 mL of freshly mixed complex-forming reagent. Let the solution stand at room temperature for 10 min

2. Add 0.1mL of diluted Folin reagent, using a vortex mixer, and let the mixture stand at room temperature for 30-60 min (do not exceed 60 min)

3. Read the absorbance at 750 nm if the protein concentration was below 500 ug/mL or at 550 nm if the protein concentration was between 100 and 2000 ug/mL.

4. Plot a standard curve of absorbance as a function of initial protein concentration and use it to determine the unknown protein concentrations.

Notes

1. If the sample is available as a precipitate, then dissolve the precipitate in 2N NaOH.

2. Peterson has described a precipitation step that allows the separation of the protein sample from interfering substances and also consequently concentrates the protein sample, allowing the determination of proteins in dilute solution. Peterson's precipitation step is as follows:
       a. Add 0.1 mL of  0.15% deoxycholate to 1.0 mL of protein sample.
       b. Vortex, and stand at room temperature for 10 min.
       c. Add 0.1 mL of 72% TCA, vortex, and centrifuge at 10000 rpm for 30 min.
       d. Decant the supernatant and then dissolve the pellet in 2N NaOH.

3. The reaction is very pH-dependent, and it is therefore important to maintain the pH between 10 and 10.5. Take care, therefore, when analyzing samples that are in strong buffer outside this range.

4. The incubation period is not critical and can vary from 10 min to several hours without affecting the final absorbance.

5. The Vortex step is critical for obtaining reproducible results. The Folin reagent is only reactive for a short time under these alkaline conditions, being unstable in alkali, and great care should therefore be taken to ensure thorough mixing.

6. The assay is not linear at higher concentrations. Ensure, therefore, that you are analyzing your sample on the linear portion of the calibration curve.

7. A set of standards is needed with each group of assays, preferably in duplicate. Duplicate or triplicate unknowns are recommended.

8. One disadvantage of the Lowry method is the fact that a range of substances interfere with this assay, including buffers, drugs, nucleic acids, and sugars. In many cases, the effects of these agents can be minimized by diluting them out, assuming that the protein concentration is sufficiently high to still be detected after dilution. When interfering compounds are involved, it is, of course, important to run an appropriate blank. Interference caused by detergents, sucrose, and EDTA can be eliminated by the addition of SDS. The best alternative in this case is to do Lowry-TCA (see Protein Precipitation Protocols) or Peterson.

9. Modifications to this basic assay have been reported that increase the sensitivity of the reaction. If the Folin reagent is added in two portions, vortexing between each addition, a 20% increase in sensitivity is achieved. The addition of dithiothreitol 3 min after the addition of the Folin reagent increases the sensitivity by 50%.

10. The amount of color produced in this assay by any given protein (or mixture of proteins) is dependent on the amino acid composition of the protein(s) (see Introduction). Therefore, two different proteins, each for example at concentrations of 1 mg/mL, can give different color yields in this assay. It must be appreciated, therefore, that using BSA (or any other protein for that matter) as a standard only gives an approximate measure of the protein concentration. The only time when this method gives an absolute value for protein concentration is when the protein being analyzed is also used to construct the standard curve. The most accurate way to determine the concentration of any protein solution is amino acid analysis.

Interfering Reagents for Lowry

Many detergents, Urea, Guanidine HCl, high sucrose, Ammonium sulphate, >0.1M TrisHCl, >1MNa Acetate or Na Phosphate, EDTA, reducing agents, etc.
 
 
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Dr. Mario Lebendiker The Protein Purification Facility
The Wolfson Centre for Applied Structural Biology,    The Hebrew University of Jerusalem
mariol@mail.ls.huji.ac.il  Tel: 972-2-6586920  

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