Assays Lowry method

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 Then 10 ml of sample were withdrawn each 24 h during fermentation and immediately filtered through Millipore membranes of 0.45 pm pore size these cell-free filtrates were used for enzymatic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... 

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... 

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. 

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. 

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. 

Assay each of the fractions preserved in steps 10-5 to 10-38 for protein concentration and enzyme activity. Use the Lowry method described in Chapter 2 for protein determinations. Use the enzyme assay procedures described in steps 10-52 to 10-57 to determine enzyme activity. 

Unless otherwise indicated transhydrogenase activity was assayed by reduction of AcPyAD by NADPH and protein was determined by the Lowry method... 

Total protein in an unknown sample was estimated using the Lowry and Bradford assays. Results were 33 2 pg/mL from the Lowry assay and 21 1 pg/mL from the Bradford assay, using stock solutions of BSA as standards in each assay. The unknown sample was then thoroughly oxygenated, and the assays were repeated. The Lowry method yielded 22 1 pg/mL, while the Bradford results did not change. Why did the results of the Lowry assay decrease by 33% ... 

Spectrophotometric analyses are the most common method to characterize proteins. TTie use of ultraviolet-visible (UV-VIS) spectroscopy is t rpically used for the determination of protein concentration by using either a dye-binding assay (e.g., the Bradford or Lowry method) or by determining the absorption of a solution of protein at one or more wavelengths in the near UVregion (260-280 nm). Another spectroscopic method used in the early-phase characterization of biopharmaceuticals is CD. 

Protein Assay. Quantitative protein assays were performed by the Lowry method (IV). Protein in column eluates was monitored at 280... 

The supernatants were concentrated with CF 25 cone, washed twice with 1.0 ml buffer, and reacted with a reaction mixture containing 0.5 n mol/I hypoxanthine at 37°C for 30 minutes. Uric acid product was determined by the rate of increased absorption of 293 nm at 37°C measured in a Gilford 2400 type recording spectrophotometer. Protein concentration of the specimens was determined by the Lowry method. Urinary oxypurines were assayed by HPLC as follows. One to two ml of urine samples from 24 hours total excretion were diluted 5-10 times with 0.9% NaCl and filtrated by a 0.45 pm Millipore filter. Three quarters of ml of this sample with 0.25 ml of 0.1 N NaOH and 1.0 ml of ethyo-acetate n- Butanol =2 1 solution were mixed for one minute. 

Autolytic assays were undertaken according to the method described by Morrissey et al. (1993) with the following changes. A 3 g surimi gel sample was finely chopped with a razor blade and incubated at 55°C for 1 hr. Autolysis was stopped by adding 27 mL 5% cold trichloroacetic acid (TCA), incubating the mixture at 4" C and centrifuging at 6100 x g for 15 min. The supernatant was analyzed for oligopeptide content by the Lowry assay (Lowry et al, 1951) and expressed as mmoles of tyrosine released. 

The Lowry and BCA methods are related, in that the first step of the assay is the reduction of copper ion, Cu to Cu, by protein amides under alkaline conditions. In the Lowry method the reduced copper— and to a lesser extent some protein side chains— react with the Folin-Ciocalteu reagent (phosphomolybdate/pho-sphotungstate) to produce a blue color. In the BCA assay the BCA complexes with the Cu, which absorbs strongly at 562 nm. Although both the Lowry and BCA methods must be carefully timed and are subject to interference from buffer components, the BCA method is less susceptible to interference, especially by detergents. 

Adsorption isotherms of jff-lactoglobulin on polystyrene latex particles were studied by Mackie et al for pH values of 4.65,12, and 9.0. The latex and attached protein were then removed by centrifugation, and the concentration of protein remaining in solution was calculated by assaying the supernatant by the Lowry method. 

Folin-Ciocalteau or Lowry method While the biuret method is sensitive in the range 0.5 to 2.5 mg protein per assay, the Lowry method is 1 to 2 orders of magnitude more sensitive (5 to 150 pg). The main disadvantage of the Lowry method is the number of interfering substances these include ammonium sulfate, thiol reagents, sucrose, EDTA, Tris, and Triton X-100. 

The final colour in the Lowry method is a result of two reactions. The first is a small contribution from the biuret reaction of protein with copper ions in alkali solution. The second results from peptide-bound copper ions facilitating the reduction of the phos-phomolybdic-tungstic acid (the Folin reagent) which gives rise to a number of reduced species with a characteristic blue colour. The amino acid residues which are involved in the reaction are tryptophan and tyrosine as well as cysteine, cystine and histidine. The amount of colour produced varies slightly with different proteins. In this respect it is a less-reliable assay than the biuret method, but it is more reliable than the absorbance method since A280 may include contribution from other species, and also the absorption of a given residue is dependent on its environment within the protein. 

Isocitric Lyase Ten DAP plants were sprayed with G-ME (5 /tM), GA3-ME (10 /tM), and G-ME + GA -ME (5, 10 /tM, IL actiyity was assayed according to Doig et al. (1975). Protein was assayed by the Lowry method (1951). Data were analyzed as discussed above. Plants were harvested at 7 days after spraying. Rve replications/treatment. 

Alkaline phosphatase (ALP) activity ALP activity of the supematarrt was meastrred using an ALP B-Test WAKO kit (Wako, Japan) based on the Bessey-Lowry method. ALP activity was normalized by samples total protein production measured with a Bicinchoninic Acid Assay (BCA, BCA Protein Assay KiL Pierce Biotechnology, U.S. A.). 

In this method the keyhole limpet haemoglobin conjugate was prepared as follows Keyhole limpet haemocyanin (KLH, Calbiochem, La Jolia, CA) and bovine serum albumin (BSA, BDH Chemicals) were coupled to the adduct (2), derived from 6-bromohexanoic acid and monoquat (3), via a carbodiimide reaction, as reported previously by Niewola et al. [184], The resulting conjugates contained 662mol of Paraquat per mole of KLH and 15mol of Paraquat per mole of 6-bromohexanoic acid. The amount of Paraquat bound to the protein was determined by spectrophotometric dithionite assay for Paraquat and the protein concentration was established by a standard Lowry test. 

Epoxide hydratase activity, with JH-benzo(a)pyrene 4,5-oxide as substrate, was assayed by the thin-layer chromatographic procedure of Jerina et al. (15). The protein content of microsomal and whole homogenate preparations was determined according to Lowry et al. (16), using bovine serum albumin as the standard, and microsomal cytochrome P-450 content was assayed by the method of Omura and Sato (17) on an Aminco DW-2A spectrophotometer. 

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