Measurement of protein

The assay result of coelenterazine described in Section C5.1 includes the amount of the protein-bound form of coelenterazine in addition to free coelenterazine. The assay of only the protein-bound form, or only the free coelenterazine, is complicated because the protein-bound form tends to liberate free coelenterazine by various stimuli, not only by Ca2+. An example of the measurement of protein-bound coelenterazine is given by Shimomura and Johnson (1979b). 

Aucamp, J.P., Cosme, A.M., Lye, G.J. andDalby, P.A. (2005) High-throughput measurement of protein stability in microtiter plates. Biotechnology and Bioengineering, 89, 599-607. 

Li, E., Placone, J., Merzlyakov, M. and Hristova, K. (2008). Quantitative measurements of protein interactions in a crowded cellular environment. Anal. Chem. 80, 5976-85. 

Figure 8.2 Design of protein-embedding barcode is depicted in (a) five thin layers of matrix (the thicker lines) coated with variable concentration of tested protein (thinner lines located above the matrix), (b) A FFPE tissue section of bladder cancer IHC-stained by monoclonal antibody to E-cadherine showing variable intensity of positive staining results which is compared with a protein-embedding bar code as designed in this chapter. Using computer-assisted image analysis with a special software, an automatic quantitative measurement of protein is performed. See color insert.

For the MSn measurement of proteins, that is, the measurement of peptides, one has to denature and reduce proteins in the tissue samples, followed by enzyme digestion. Therefore, protein samples should be treated with trypsin after membrane transfer. Trypsin can be attached to the membrane and can be performed in the same steps as in the matrix coating method. 

Antibodies have been used extensively as diagnostic tools in many different formats, especially for measurement of protein and cytokine abundances. Applications of antigen arrays can be found in reverse immunoassays for detection of allergens and autoimmune antibodies. 

Shumaker-Parry JS, Aebersold R, Campbell CT (2004) Parallel, quantitative measurement of protein binding to a 120-element double-stranded DNA array in real time using surface plasmon resonance microscopy. Anal Chem 76 2071-2082... 

Simply on the basis of the normal composition of marine organisms, we would expect proteins and peptides to be normal constituents of the dissolved organic carbon in seawater. While free amino acids might be expected as products of enzymic hydrolysis of proteins, the rapid uptake of these compounds by bacteria would lead us to expect that free amino acids would normally constitute a minor part of the dissolved organic pool. This is precisely what we do find the concentration of free amino acids seldom exceeds 150 xg/l in the open ocean. It would be expected that the concentration of combined amino acids would be many times as great. There have been relatively few measurements of proteins and peptides, and most of the measurements were obtained by measuring the free amino acids before and after a hydrolysis step. Representative methods of this type have been described [245-259]. Since these methods are basically free amino acid methods, they will be discussed next in conjunction with those methods. 

The older methods for the measurement of protein in natural waters usually depended upon the presence of aromatic amino acids in the protein, and calculated total protein on the basis of an average tyrosine, tryptophan, or phenylalanine content. A method representative of this type was the Folin reagent method published by Debeika et al. [281]. While these methods were useful in fresh water and in some coastal regions, they were not sensitive enough for the lower concentrations to be found in oceanic waters. 

Temperature consistency between measurements performed on different spectrometers is particularly critical for accurate interpretation of the data (see Refs. [19, 20] for post-acquisition temperature consistency tests). However, temperature control and equalization are also important for the combined analysis of T1, T2, and NOE data measured on the same spectrometer, because of the possible temperature differences between these measurements. Fig. 12.1 illustrates the sensitivity of relaxation parameters to temperature variations. Accurate measurement of protein dynamics requires that all experiments be done at the same temperature. To improve temperature consistency between Tlr T2, and... 

R. J. Cherry, Measurement of protein rotational diffusion in membranes by flash photolysis, Methods Enzymol. L1X, 47-61 (1978). 

Measurement of Protein Mobility Within Cell Membranes... 

E. Bouveresse, D.L. Massart and P. Dardenne, Calibration transfer across near-infrared spectroscopic instruments using Shenk s algorithm effects of different standardisation samples. Anal. Chim. Acta, 297, 405 16 (1994). B.G. Osborne and T. Feam, Collaborative evaluation of universal cahbrations for the measurement of protein and moisture in flour by near-infrared reflectance, /. Food TechnoL, 18, 453 60 (1983). 

The ALIS quench method for dissociation rate measurement uses little protein and requires no biochemical assay for its implementation, yet the method readily yields quantitative values for the dissociation rates of the protein-ligand complexes. The technique can be used with pools of ligands to provide a quantitative rank ordering of the dissociation rates of all the components of the mixture. Since it is not necessary to know the exact concentrations of the ligands under study, the dissociation rate assessment can be performed using impure compounds, such as unpurified compound mixtures derived from combinatorial chemistry synthesis. The method does not require a foreknowledge of active protein concentration to measure and rank ligands based on their rates of dissociation. As such, the technique is self-contained and does not rely upon an external measure of protein activity as one of its input parameters. 

Powell, K.D. Fitzgerald, M.C. Measurements of protein stability by H/D exchange and matrix-assisted laser desorption/ionization mass spectrometry using picomoles of material. Ancd. Chem. 2001, 73, 3300-3304. 

JP Hummel, WJ Dreyer. Measurement of protein-binding phenomena by gel filtration. Biochem Biophys Acta 63 530-532, 1962. 

Soares, J.C., Chen, G., Dippold, C.S., Wells, K.F., Frank, E., Kupfer, D.J., Manji, H.K., and Mallinger, A.G. (2000) Concurrent measures of protein kinase C and phosphoinositides in lithium-treated bipolar patients and healthy individuals a preliminary study. Psychiatry Res 95 109-118. 

Figure 7.9. A possible metrological traceability chain for the result of a measurement of protein in a sample of grain. aTris = 2-amino-2-hydroxymethyl-1,3-propanediol b Dumas apparatus is calibrated using tris CRM and grain samples are certified in an interlaboratory study cthe master instruments measure grain samples to act as the grower s calibrator for field measurements.

Bloustine, J., Berejnov, V., Fraden, S. (2003). measurements of protein-protein interactions by size exclusion chromatography. Biophysic. J., 85, 2619-2623. 

Tessier, P.M., Lenhoff, A.M., Sandler, S.I. (2002). Rapid measurement of protein osmotic second virial coefficients by self-interaction chromatography. Biophysical Journal, 82, 1620-1631. 

For gelatlon/coagulatlon Investigations to be useful measures of protein functionality. It Is essential that the conditions be... 

Zone electrophoresis is mostly used for biological applications. Peptide separation and the measurement of protein fractions from blood serum (proteinogram of albumin and o-, (3- and 7-globulins) are among the better known applications. This TLC for biochemists is useful for the separation of polysaccharides, nucleic acids (for DNA sequencing), proteins and other colloidal species. 

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