Lowry-protein method

A method that has been the standard of choice for many years is the Lowry procedure. This method uses Cn ions along with Folin-Ciocalteau reagent, a combination of phosphomolybdic and phosphotnngstic acid complexes that react with Cn. Cn is generated from Cn by readily oxidizable protein components, such as cysteine or the phenols and indoles of tyrosine and tryptophan. Although the precise chemistry of the Lowry method remains uncertain, the Cn reaction with the Folin reagent gives intensely colored products measurable spectrophotometrically. 

Experiments in 500 ml Erlenmeyer flasks and Fernbach flasks contained 200 ml and 1 L of EPl and EP2 medium respectively. Inocuia added to these cultures was 2 ml of spore suspension (5.0 optical density at 540 nm) for each 100 ml EP medium. All cultures were grown at 37°C in a shaking incubator (New Brunswik Sci. Co., USA), at 200 rpm. Then 10 ml of sample were withdrawn each 24 h during fermentation and immediately filtered through Millipore membranes of 0.45 pm pore size these cell-free filtrates were used for enzymatic assays and extracellular protein determinations by the Lowry method (14). Experiments in the 14 L fermentor (Microgen Fermentor New Brunswik Sci. Co., USA) were carried with lOL of fermentation medium EP2 and inoculum added was IL of mycelium grown 24 h in... 

Protein concentration was determined in cell free filtrates, according to the Lowry method (14) with bovine seric albumin (BSA) as standard. 

The specimens were analyzed at spectrophotometer at 280 nm. Preparations were purified from salts by dialysis. Protein concentrations in the initial and purified enzyme preparations were determined by Lowry method [6]. 

Lowry method uses a combination of the Biuret copper-based reagent and the Folin-Ciocalteau reagent, which contains phosphomolybdic-phosphotungstic acid. Reagents react with protein, yielding a blue colour that displays an absorbance maximum at 750 nm... 

Bradford reagent contains the dye Coomassie blue G-250 in an acidic solution. The dye binds to protein, yielding a blue colour that absorbs maximally at 595 nm Copper-containing reagent that, when reduced by protein, reacts with bicinchonic acid yielding a complex that displays an absorbance maximum at 562 nm Essentially involves initial precipitation of protein out of solution by addition of trichloroacetic acid. The protein precipitate is redissolved in NaOH and the Lowry method of protein determination is then performed Interaction of silver with protein - very sensitive method... 

For activity assays, proteinase solutions were made fresh daily (10 mg freeze-dried solids in 1 ml pH 10.0 phosphate buffer, 0.1 M). Two ml of the proteinase, 0.5 ml of substrate (azocasein or other proteins in pH 10 buffer), 0.3 ml of 0.1% EDTA, and deionized water were made up to a volume of 3.5 ml. Reaction tubes were incubated in a 40°C water bath for 1 hr, then the reaction was stopped by addition of 1 ml 5% TCA. After removal of the precipitated proteins by centrifugation, absorbance was read at 366 nm (for azocasein) or by the Lowry method (15) for other... 

This is a modification of the Lowry method involving a dye-binding step. The copper-protein complex that forms the basis of the biuret and the Lowry methods can be chelated by bicinchoninic acid to produce a very stable complex with a strong absorption maximum at 562 nm. 

It is said to suffer from less interference effects than the Lowry method and is capable of detecting protein levels as low as 10 /xg. The presence of lipids does interfere with the assay and modification of the technique is required if detergents are present. The reagent is only stable for a short time but if the copper sulphate is only added prior to use the stock reagents keep indefinitely. 

If analytical methods are at the heart of biopharmaceutical development and manufacturing, then protein concentration methods are the workhorse assays. A time and motion study of the discovery, development, and manufacture of a protein-based product would probably confirm the most frequently performed assay to be protein concentration. In the 1940s Oliver H. Lowry developed the Lowry method while attempting to detect miniscule amounts of substances in blood. In 1951 his method was published in the Journal of Biological Chemistry. In 1996 the Institute for Scientific Information (ISI) reported that this article had been cited almost a quarter of a million times, making it the most cited research article in history. This statistic reveals the ubiquity of protein measurement assays and the resilience of an assay developed over 60 years ago. The Lowry method remains one of the most popular colorimetric protein assays in biopharmaceutical development, although many alternative assays now exist. 

The BCA method is simpler than the Lowry method and relies on the same redox reaction. The target protein is treated with alkaline cupric sulfate in the presence of tartrate, which results in the reduction of the cupric ion to cuprous by the protein. The cuprous ion is then treated with bicichoninic acid (BCA) and two... 

Dilute sarcolemmal vesicles from Protocol 5.3.2 to about 1 mg pro-tein/ml with Soln. A (for protein concentration determination the Lowry method is recommended). Add 1 pi of Soln. B per 100 pi SL dilution, vortex and put on ice. Pipet the probes in triplicates into Eppendorf tubes according to Table 5.6. Label the ouabain-containing tubes with those without ouabain should be signed... 

Cell pellets are thawed at room temperature. Then each tube is sonicated twice for at least 10 s, with cooling on ice between each 10-s burst. The protein concentration is determined by a routine method (i.e., the Lowry method) and the result recorded for each cell pellet. 

Purity was confirmed by gel-filtration using a HPLC column packed with Asahipak GS-520HQ and elution with 100 mM sodium phosphate buffer containing 300 mM sodium chloride (pH 6.7). The content of total protein, total sugars, uronic acids, sulfates, nucleic acids, phosphate or fatty acids was assayed by the BCA [32] and Lowry method [33], the phenol-sulfuric acid method [34], the Blumenkrantz method [35], nephelometry [36], absorption at 260 nm, the Bartlett method [37] and the GLC method after methyl-esterification [38], respectively. 

This unit describes four of the most commonly used total protein assay methods. Three of the four are copper-based assays to quantitate total protein the Lowry method (see Basic Protocol 1 and Alternate Protocols 1 and 2), the bicinchoninic acid assay (BCA see Basic Protocol 2 and Alternate Protocols 3 and 4), and the biuret method (see Basic Protocol 3 and Alternate Protocol 5). The fourth is the Coomassie dye binding or Bradford assay (see Basic Protocol 4 and Alternate Protocols 6 and 7), which is included as a simple and sensitive assay, although it sometimes gives a variable response depending on how well or how poorly the protein binds the dye in acidic pH. A protein assay method should be chosen based on the sensitivity and accuracy of method as well as the condition of the sample to be analyzed. 

For greatest accuracy of the estimates of the total protein concentration in unknown samples, it is essential to include a standard curve in each run. This is particularly true for the protein assay methods that produce nonlinear standard curves (e.g., Lowry method, Coomassie dye-binding method). The decision about the number of standards used to define the standard curve and the number of replicates to be done on each standard depends upon the degree of nonlinearity in the standard curve and the degree of accuracy required of the results. In general, fewer points are needed to construct a standard curve if the color response curve is linear. For assays done in test tubes, duplicates are sufficient however, triplicates are recommended for assays performed in microtiter plates due to the increased error associated with microtiter plates and microtiter plate readers. 

Protein tested Ratios obtained with the BCA method Ratios obtained with the Coomassie plus method Ratios obtained with the modified Lowry method... 

Peterson, G.L. 1977. A simplification of the protein assay method of Lowry, et al. Which is more generally applicable Anal. Biochem. 83 346-356. 

Peterson, G.L. 1979. Review of the Folin phenol protein quantitation method of Lowry, Rose-brough, Farr and Randall. Anal. Biochem. 100 201-220. 

Additional reagents and equipment for Kjeldahl, Lowry, or other protein determination method (unitbi.i)... 

Fig. 1 Impact of sodium azide (NaN3 1 mg/ml) on total protein concentration in urine determined by the Lowry method...

Fig. 3 Determination of total protein concentration (Lowry method) A, standard albumin A+G, standard BIORAD -70% albumin +30% globulin...

Table 1 Six methods for the determination of total protein concentration in human urine were compared the Lowry method, biuret method, methods using the dyes Commassie Brilliant Blue (G250), Ponceau-S, pyrogallol red and the turbidity method by Exton...

AOD was immobilized on aminopropyl glass beads that were first treated with 2.5% glutaraldehyde, staying in contact with the enzyme for 24 h at 30°C in a rotatory shaker incubator at 50 rpm. The protein concentration in buffer solution was quantified before (PCBI) and after (PCAI) the immobilization proceeding, using the Lowry method. The retention efficiency was calculated using Eq. 1. 

Xylanase was assayed using birchwood xylan as substrate. The solution of xylan and the enzyme at appropriated dilution were incubated at 75°C for 3 min, and the reducing sugar was determined by the dinitrosali-cylic acid procedure (12) with xylose as standard. The released color development was measured spectrophotometrically at 540 nm. One unit of enzyme activity was defined as 1 pmol of reducing sugar released 1 min under the described assay conditions. Protein concentration was measured by the Lowry method (13) using bovine serum albumin as standard. 

Porcine brains, which were obtained within 30 min after death of the animals from a local slaughterhouse, were used to prepare receptor-rich membranes. The brains were immediately frozen in liquid N2 50 g brain per 200 mL ice-cold buffer (0.32 M sucrose, 10 mM potassium phosphate buffer, pH 7.0, 1 mM EDTA) were homogenized twice for 15 sec in a blender and then for 1 min with an ultraturrax. The homogenate was centrifuged three times for 15 min at 1.400 g and 4°C to separate cellular debris. The supernatant was spun down at 100.000 g for 60 min. The resulting pellet was resuspended in buffer (as above but without sucrose). Aliquots were stored frozen at -80°C. Protein content was determined by the Lowry method, using bovine serum albumin as a standard.32 35... 

Male Wistar-Kyoto rats weighing 150 g receive either continuous administration of the test drug by an osmotic pump (ALZA Co., Palo Alto, USA) or saline. Twenty-four hours later, the rats are injected with 1 ml of nephrotoxic serum. At 9, 12, and 14 days, urine samples are collected and urinary protein levels are measured using the Lowry method. At 14 days the rats are sacrificed under ether anesthesia, and both kidneys are removed. Portions of these tissues are processed for light microscopy, immunofluorescence staining and immunoperoxidase staining. 

---