This is a cached page for the URL (http://www.animal.ufl.edu/hansen/protocols/lowry.htm). To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
Lowry Protein Assay - University of Florida
LOWRY PROTEIN ASSAY

P.J. Hansen
Dept. of Animal Sciences, University of Florida

More Techniques     |  P.J. Hansen Home Page   |   UF Animal Sciences Home Page



The Lowry procedure is one of the most venerable and widely-used protein assays, being first described in 1951 [Lowry et al., J. Biol. Chem. 193: 265-275 (1951)]. Under alkaline conditions, copper complexes with protein. When folin phenol reagent (phospho-molybdic-phosphotungstic reagent) is added, the Folin-phenol reagent binds to the protein. Bound reagent is slowly reduced and changes color from yellow to blue.

While widely used, the Lowry procedure is less preferable an assay than some other protein assays since it is more subject to interference by a wide variety of chemicals. Among the chemicals reported to interfere with the Lowry procedure are barbital, CAPS, cesium chloride, citrate, cysteine, diethanolamine, dithiothreitol, EDTA, EGTA, HEPES, mercaptoethanol, Nonidet P-40, phenol, polyvinyl pyrrolidone, sodium deoxycholate, sodium salicylate, thimerosol, Tricine, TRIS and Triton X-100.

There is also much protein-to-protein variation in the intensity of color development. Ideally, the standard should be similar to the unknown. For example, if one is measuring IgG concentrations, an immunoglobulin standard would be ideal. For serum, use bovine serum albumin as a standard since albumin is a major component of serum.

An easy and accurate alternative, based on the binding of protein to Coomassie Blue G-250 dye, is the Bradford procedure. In addition, a modification of the Lowry procedure exists based on use of bicinchoninic acid (BCA) in place of the Folin-phenol reagent [Smith et al., Anal Biochem.150, 76-85 (1985)]. The BCA is less prone to interference than the Lowry procedure and is more sensitive.

Stock Solutions

Lowry A: 2% Na2CO3 in 0.1 M NaOH
Lowry B: 1% CuSO4 in diH2O
Lowry C: 2% sodium potassium tartrate (NaKC4H4O6 4H2O)

Reagents

Lowry stock reagent
    49 ml Lowry A
    0.5 ml Lowry B
    0.5 ml Lowry C

Folin's Reagent: Phenol reagent - 2N (Folin - Ciocalteau reagent). Dilute 1:1 in diH2O before use.

Standard: Bovine serum albumin (BSA), lysozyme or other protein. If the standard is weighed out before use, make sure that the protein preparation used to prepare the standard is salt-free to avoid inaccurate results. The standard should be dissolved at a concentration of 1 mg/ml in a buffer similar to the solution the unknown is in (use PBS for biological samples)and diluted as follows:
 

micrograms protein standard microliters standard microliters buffer
0 0 100
10 10 90
20 20 80
30 30 70
50 50 50
75 75 25
100 100 0

Procedure

1) Add 100 ul of sample (sample + buffer = 100 ul) per tube.

2) Add 1.0 ml of Lowry stock reagent to each tube.

3) Incubate 30 min at room temperature.

4) Add 100 ml of Folin's reagent to each tube.

5) Incubate 30 min at room temperature.

6) Read in a spectrophotometer at 595 nm.
 
 



produced 7-90, html prepared 02-21-01.  For questions, contact  Peter J. Hansen

©1990 and 2001 P.J. Hansen