Assays for proteins

Figure 2. Gel filtration. The dry residue obtained after ammonium sulfate precipitation was redissolved in 50 mM phosphate buffer, pH 7.4 0.15 M NaCl 0.013 % sodium azide, which was loaded on a Superdex 75HR1030 column equilibrated with the same buffer. Elution was downward flow (0.15 ml/min) and 0.25 ml fractions were collected. The fractions were assayed for protein content (— ) and PNL activity (- - ).

SDS-PAGE was performed by the method of Laemmli [17]. The methods for native PAGE, isoelectric focussing, detection of esterase activity in electrophoresis gels, and assays for protein glycosylation have been described elsewhere [5]. 

Sphaeroplasts were prepared by slight modifications to published methods [12,13]. Lysis of sphaeroplasts was effected by a combination of osmotic lysis and gentle mechanical disruption [14]. Discontinuous sucrose-density gradients were constructed and fractions were then assayed for protein, PG and marker enzymes for different organelles. 

Fig. 7. Coomassie Brilliant Blue R-250 (I) is used to stain proteins, e.g., after gel-electrophoretic separation, its derivative G-250 (II) is applied in the Bradford assay for protein quantification... 

Three animal procedures (protein efficiency ratio, net protein ratio, and protein digestibility) were used to evaluate protein quality. The AOAC (13) animal assay for protein efficiency ratio... 

Lucas developed an assay for protein A contamination.16 Protein A from S. aureus is commonly used for purifying immunoglobulin because of its specificity for binding immunoglobulins of several species. For an assay to accurately quantitate amounts of contaminating protein A, it must be able to measure it in the presence of a large excess of another protein with which it interacts (because... 

Heinicke, E. Kumar, U. Munoz, D. G. Quantitative dot-blot assay for proteins using enhanced chemiluminescence. J. Immunol. Methods 1992,152(2), 227-236. 

Measurement of DNA solution using bisbenzimidazole dye (Hoechst 33258). http //wwwmamgen tubitakgovtr/taylan/protocols/spot—-00 txt EtBr spot test for DNA and RNA analysis http //www swmed edu/home pages/personal/davie/Protein html Bradford plate assay for protein... 

Beyer, R.E. 1983. A rapid biuret assay for protein of whole fatty tissues. Anal. Biochem. 129 483-485. 

Watters, C. 1978. A one-step biuret assay for protein in the presence of detergent. Anal. Biochem. 88 695-698. 

Assay for protein-substrate hydrolysis as described (see Basic Protocol 3, steps 3 to... 

Diazonium ions react rapidly with histidyl, lysyl, and tyrosyl residues to form mono- and di-azo derivatives, and much more slowly with arginyl, cysteinyl, and tryptophanyl residues.18 The lack of specificity of the reaction can create problems in interpreting results, particularly if critical residues are modified. The azo-proteins formed may be readily purified, and are intensely colored this property may permit quantitation of the extent of the reaction by measurement of the absorbance. Not uncommonly, however, the products of the reaction are of a dark-brownish color that interferes with many of the standard assays for protein and carbohydrate. 

Thymidine Uptake Studies. Tritiated thymidine (52 mCi/ anole 0.1 /rCi/ml) was added to cells for 60 min at 37°C. Cells were then washed with PBS, incubated at 4°C for 15 min in the presence of ice cold 5% TCA, rinsed with TCA and scraped from the flasks with a rubber policeman. Cells were again washed with PBS, and solubilized in 0.1N NaOH overnight. Aliquots were assayed for protein (62) and radioactivity (scintillation fluid 100 ml Biosolve (Beckman, Fullerton, CA.), 7g of PPO and 0.6 g of POPOP per liter of toluene). 

JE Casnellie. Assay for protein kinases using peptides with basic residues for phos-phocellulose binding. Meth Enzymol 200 115-120, 1991. 

Cohen, C.B., Chin-Dixon, E., Jeong, S., Nikiforov, T.T., A microchip-based enzyme assay for protein kinase A. Anal. Biochem. 1999, 273, 89-97. 

Anderson NL (2010) The clinical plasma proteome a survey of clinical assays for proteins in plasma and serum. Clin Chem 56 177-185... 

Koresawa, M. and T. Okabe. 2004. High-throughput screening with quantitation of ATP consumption a universal non-radioisotope, homogeneous assay for protein kinase. Assay Drug Dev. Technol. 2, 153-160. 

McCain, D.F. and Z.Y. Zhang. 2001. Assays for protein-tyrosine phosphatases. Meth. Enzymol. 345, 507-518. 

Bicinchoninic Acid (BCA) Assay. The bicin-choninic acid assay for proteins is based on the same reactions as the Folin-Ciocalteau assay. Proteins are again reacted with alkaline cupric ions to form the biuret complex, and these ions are reduced to cuprous ions by the aromatic amino acids in the proteins. In this case, however, the Cu1+ ions form a complex with bicinchoninic acid (Fig. II-6), which has an intense absorbance maximum at 562 nm. This assay shows the same variation from protein to protein as the Folin-Ciocalteau assay, but is more convenient experimentally and can be made somewhat more sensitive. 

A15. Armstrong, S. G., and Dean, R. T., A sensitive fluorometric assay for protein-bound DOPA and related products of radical-mediated protein oxidation. Redox Rep. 1, 291-298 (1995). 

Bovine serum albumin (BSA) and cyclic AMP (cAMP) are determined by a competitive binding enzyme immunoassay (315). With urease as label, an ammonia gas-sensing electrode is used to measure the amount of urease-labeled antigen bound to a double-antibody solid phase by continuously measuring the rate of ammonia produced from urea as substrate. The method yields accurate and sensitive assays for proteins (BSA less than 10 ng/mL) and antigens (cAMP less than 10 nM), with fairly good selectivity over cGMP, AMP, and GMP. 

Gu, L., Jones, A.D., and Last, R.L. LC-MS/MS assay for protein amino acids and meta-bohcally related compounds for large-scale screening of metabolic phenotypes. Anal. Chem. 2007, 79, 8067-8075. 

A combination of PPIA and microcystin immunoassay was proposed by Carmichael et al. (1999) to indicate the potential toxicity of a bloom sample and the concentration of the microcystins. A combined assay, consistent with this principle, was developed by Metcalf et al. (2001) this includes preexposure of the sample to microcystin antibodies, to make microcystins/nodularins that are present biounavailable to the subsequent addition of protein phosphatase enzyme, before assaying for protein phosphatase inhibitoiy activity. The resulting assay, termed the colorimetric immunoprotein phosphatase inhibition ass (CIPPIA), was found to be specific for microcystins and nodularins since the microcystin antibodies protect the protein phosphatase from inhibition by the toxins. Complete protection from inhibition of protein phospliatase by the antibodies indicates that the inhibition of the protein phosphatase in the sample was due to the cyanobacterial toxins. These colorimetric assays showed a good correlation with the HPLC analysis of extracts cyanobacteria. Immunoassays can also be combined with physicochemical methods such as HPLC (Zeck 2001b). In this case, the HPLC method separates the microcystins according to their hydrophobicity and the resulting fractions are analyzed by immunoassay. 

Pais, J.E., Bowers, K.E., Stoddard, A.K., and Fierke, C.A. (2005). A continuous fluorescent assay for protein prenyltransferases measuring diphosphate release. Anal Biochem 345 302-311. 

Walker, J.M. (1994b) The Bicinchonic (BCA) Assay for Protein Quantitation, Methods Md. Biol. 

To obtain an active preparation it is essential to perform all steps without delay. Furthermore, avoid keeping synaptosomes resuspended for prolonged periods of time (e.g., use a fast dye-binding assay for protein determination that gives results in 10 min or less, such as that described by Bradford, 1976). The aliquoted synaptosomal pellets should not be resuspended until immediately before being used in the experiment. 

To minimize complexity and cost, yet retain enough accuracy and precision to meet clinical demands, most commercially available assays for proteins are a compromise In those circumstances in which these are more demanding —for example, for transferring values to a manufacturer s gold standard calibrators and controls—signal level should be maximized and all steps in the assay should be performed with as high precision as possible. The criteria for such an assay have been summarized by Blirup-Jensen. Reference laboratories should also use this or similar protocols to maximize accuracy. 

Joos TO, Schrenk M, Hopfl P, et al (2000) A microarray enzyme-linked immunosorbent assay for autoimmune diagnostics. Electrophoresis 21(13) 2641-2650 Martzen MR, McCraith SM, Spinelli SL, et al (1999) A biochemical genomics approach for identifying genes by the activity of their products. Science 286(5442) 1153 -1155 Cohen CB, Chin-Dixon E, Jeong S, et al (1999) A microchip-based enzyme assay for protein kinase A. Anal Biochem 273(l) 89-97... 

---