Lowry assay, protein determination

A Biuret assay to determine protein concentration 190 A Lowry assay to determine protein concentration 191 A bidnchoninic assay to determine protein concentration 192 A dye-binding assay for the estimation of protein concentrations 193 A fluorescence assay for the estimation of protein concentrations 194... 

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 Then 10 ml of sample were withdrawn each 24 h during fermentation and immediately filtered through Millipore membranes of 0.45 pm pore size these cell-free filtrates were used for enzymatic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

Basic Protocol 1 The Lowry Protein Assay for Determination of Total Proteins B1.1.3 Alternate Protocol 1 Modified Lowry Protein Assay for Determination... 

THE LOWRY PROTEIN ASSAY FOR DETERMINATION OF TOTAL PROTEINS... 

Protein determination by the Kjeldahl method is slow and very few samples can be run at one time. The Lowry and many other protein tests are much more convenient. A Lowry assay on a test tube rack of 40 samples can be done in <2 hr. 

Enzyme source (see Note 4) Chinese hamster ovary cells (CHO-Kl, American Type Culture Collection) were transiently transfected via standard DEAE-dextran methods with an expression construct encoding the human ECE-lc protein. The medium was changed to serum-free medium the day after transfection. Two days after transfection, cells were harvested and a crude membrane fraction sedimented by ultracentrifugation (100,000g for 1 h). The pellet was suspended in assay buffer, aliquoted, and frozen at -70°C until assay. Protein concentration was determined by standard methods (e.g., Lowry or BCA). The equivalent of 3 pg total protein was used in each assay well. 

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. 

The Lowry assay [16] uses the reaction of cupric sulfate at alkaline pH in the presence of tartrate, producing a blue chromogen formed from four peptide bonds and one atom of copper. Addition of folin phenol reagent further enhances the color, with a maximum absorbance at 750 nm. The Lowry assay demonstrates the greatest sensitivity of the common protein concentration determination methods and varies only slightly when using the two common calibrators, BS A and BGG. Not surprisingly, this remains a very commonly used method. 

Disadvantages It takes 25 or 26 h until you can read your result. In addition, you have to manipulate the sample repeatedly hydrolyze, speed-vac, pipette, centrifuge, pipette, heat up. This is not only a test of your patience exactness also suffers. Hence, I fear that the Starcher assay will not be able to reach a similar number of citations as the Lowry. However, it makes the impression of being reliable, and if you need a protein determination with low protein-to-protein variation I would recommend it. 

The final resuspension should be in a volume calculated to give an appropriate protein concentration approx 100 mg of membrane protein is obtained for every gram of brain tissue prepared. After the final resuspension, a sample of the homogenate is taken for determination of protein content by Bradford or Lowry assays. 

Merino wool top was cleaned by sohxlet extraction using dichloromethane to remove fatty matters and repeated washing with cold water. Hie wool was air dried at about 40°C. The wool proteins were extracted overnight from wool by an extensive treatment with 8M Tris/urea buffer containing (SO mM) dithiothreitol as reduction chemical at pH 9.3 and 25 °C. The reaction was stopped using 20% iodoacetamide. The wool proteins were dialysed against distilled water for 4 days. The protein content of the enzymes and the wool hydrolysate were determined using the Lowry assay [6]. 

It is important to be able to determine which chromatography column fractions contain polysaccharides and, specifically, which fractions contain hexosyl, pentosyl, or uronosyl residues. It is also important to detect in the fractions the presence of proteins and the presence of the specific amino acid characteristic of wall proteins, hydroxyproline. The detection of these substances is carried out by facile and sensitive colorimetric procedures. Although these reactions are not discussed here, the most frequently used colorimetric assays in our laboratory are the anthrone assay for detection of hexosyl residues 50), the orcinol assay for detecting pentosyl residues 50), the m-hydroxy-diphenyl assay for detection of uronosyl residues 35), the Lowry assay for detection of proteins 90), and the Kivirikko and Liesmaa assay for the detection of hydroxyprolyl residues 76). 

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. 

COMPOUNDING OF ERRORS. Data collected in an experiment seldom involves a single operation, a single adjustment, or a single experimental determination. For example, in studies of an enzyme-catalyzed reaction, one must separately prepare stock solutions of enzyme and substrate, one must then mix these and other components to arrive at desired assay concentrations, followed by spectrophotometric determinations of reaction rates. A Lowry determination of protein or enzyme concentration has its own error, as does the spectrophotometric determination of ATP that is based on a known molar absorptivity. All operations are subject to error, and the error for the entire set of operations performed in the course of an experiment is said to involve the compounding of errors. In some circumstances, the experimenter may want to conduct an error analysis to assess the contributions of statistical uncertainties arising in component operations to the error of the entire set of operations. Knowledge of standard deviations from component operations can also be utilized to estimate the overall experimental error. 

To further characterize the drip solution, measure its protein/solids content, e.g., by performing a colorimetric assay for total protein (e.g., Lowry unitbu) and possibly analyzing by electrophoresis (unitB3.i) or chromatography (unitB4.2) to determine which proteins are actually lost in the drip solution. 

The assays for the enzyme synthesis of sulfogalactosyl-glycerolipid, sulfatides and galactocerebrosides were carried out as previously described respectively by Subba Rao, et al. (28) Sarlieve, et al. 05, 29), and Neskovic, et al. (30). The assay for 2, 3 cyclic nucleotide phosphohydrolase was performed according to the method of Prohaska, et al. (31). EL coli. alkaline phosphatase type III-S, 2, 3 -cAMP, and sodium deoxycholate were obtained from Sigma (St. Louis, Mo.). Protein was determined by the method of Lowry, et al. (32) with crystalline bovine serum albumin as the standard. 

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. 

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

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