Assays Biuret method

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. 

Other methods Although other methods have been used for total serum protein quantification, none is more convenient or practical than the biuret method. These methods are reviewed here predominantly as background information for the assay of total protein in urine or CSF. They are dependent on four general properties of proteins. 

Methods of analysis. The total protein concentration of crude solutions, including cellular proteins, was determin by the biuret method (72). With dilute xanthan solutions, this method was not sensitive enough, and a spectrofluorometric assay was carried out. This method consisted of an acid hydrolysis of the samples followed by derivatization with o-phthaldialdehyde and HPLC analysis on a Cl8 reverse column of the amino acids released, using spectrofiuorescence as the detection nKxie. 

Various techniques Glucose was assayed by glucose oxidase method according to Boehringer Carbohydrate metabolism was examined by means of the iv-tolbutamide test calculation was performed according to Lange and Quick (lO) Protein was determined by the biuret method according to Sols (11) ... 

Protein was determined by the method of Lowry, using a calibration curve which had been obtained with a TK solution standardized by the Biuret method. TPP was determined by radioactivity measurements. The transketolase assay was carried out in the presence and absence of added thiamine diphosphate/Mg++. A stimulation of 13 % of enzymatic activity was found when the coenzyme was added, indicating a loss of bound coenzyme upon gel filtration. Assuming a linear relationship between enzymatic activity and thiamine diphosphate molecules bound to apotransketolase the value found for thiamine diphosphate was corrected by these 13 %. 

Microsomes from normal and host liver and tumoral tissues were isolated by differential centrifugation as described by Brenner Peluffo (1966). Microsomal protein was determined by the biuret method (Gornall et al., 1949). The assay for long chain acyl CoA synthetase activity was based on the method of Suzue Marcel (1972). 

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. 

Soluble protein content was determined by the biuret method [11] after centrifugation of the sample at ISOOOrpm for 20 minutes. Lysozyme activity was determined by following the rate of lysis of a suspension of Micrococcus lysodeikticus cells [12]. PAA remaining in solution was assayed for turbidi-metricity by a method described below. 

For the assay of ephedrine in the total alkaloids a colorimetric method based on the biuret reaction was used by Feng and Read and is described in detail by Feng. Krishna and Chose separated ephedrine and iji-ephedrine by treating the dry mixed hydrochlorides with dry chloroform in which the ephedrine salt is virtually insoluble and the -ephedrine salt soluble. ... 

Review methods for protein assay including absorbance and colorimetric (Lowry, Biuret, Bradford, BCA). 

Many methods are available for measuring TCA-soluble peptides. Possibly the easiest is to measure the absorbance of the solution at 280 nm, as the absorbance at this wavelength is a function of the aromatic amino acid content of the solution. This approach requires a UV spectrophotometer, and the sensitivity of the assay is likely to be lower than that of some of the colorimetric assays. There are also several colorimetric peptide assays that can be applied to this type of peptidase assay, such as the Biuret, Lowry, and Bradford dye-binding methods (for comparison see Piyachomkwan and Penner, 1995). All of these methods measure a relative value rather than an absolute amount of peptide in solution. The results should thus be reported in terms of equivalents, such as BSA equivalents when using a calibration curve prepared using a BSA standard solution. 

Folin-Ciocalteu (Lowry) Assay. The quantitative Folin-Ciocalteu assay (also often called the Lowry assay ) can be applied to dried material as well as to solutions. In addition, the method is sensitive samples containing as little as 5 /Ug of protein can be analyzed readily. The color formed by the Folin-Ciocalteu reagent is thought to be caused by the reaction of protein with the alkaline copper in the reagent (as in the biuret test) and the reduction... 

We strongly recommend examination of urine immediately after collection using a microelectrode to determine pH, rather than using a dipstick. Also, for rodent urine, the Bradford method for assaying protein is preferable to biuret-based methodology (Cohen 1995). 

Many physicochemical assays are established to quantify the protein mass. It is determined by exploiting the extinction coefficient in optical density measurements or by colorimetric assays such as the Bradford, Lowry, bicinchoninic (BCA), and biuret assay [13, 14]. Albeit easy to perform, these colorimetric assays suffer from inaccuracies that are due to the use of inappropriate standards like bovine serum albumin. If relevant standards are not available, quantitative amino acid analysis [6], the (micro-)Kjeldahl nitrogen method [14, 15] or gravimetry as very accurate but time-consuming alternatives can be applied. 

As with the Biuret assays it may be necessaty to remove soluble materials that interfere with the assay. Common interfering agents include a number of common buffers (e.g. HEPES) and neutral detergents (e.g. Triton XiOO). Indeed the Folin method is notorious for the number of compounds that interfere (5, 6). One of the easiest approaches is to precipitate the protein using icecold trichloroacetic add (TCA) as described above under the Biuret assay. A number of variations on the original method have been developed to overcome interference, particularly by detergents in membrane-bound proteins (9). 

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