Quantification protein

Protein quantification is usually performed with commercially available software for spot detection and quantification. Spots are first outlined automatically and 

Matrix-Assisted Laser Desorption/lonization Time-of-Flight Mass Spectroscopy 

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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... 

There is a strong limitation in the concentration range due to the logarithmic relationship between transmission and concentration (optical densities reasonably to measure range from 0.1 to 1.5). Nevertheless, protein quantification by direct UV-measurement or after staining with dyes in the visible range is a very robust method and can be found, e.g., as a common detection mode in HPLC or other chromatographic techniques. 

Once solubilization of the membrane protein has been achieved, a reliable assay for it must exist. If the protein is an enzyme, then one must quantify the specific activity (spc. act.) of the enzyme, i.e., pmol product formed or substrate disappeared per min per mg protein. Thus, not only must the activity of the enzyme be assayed (44) but also the protein content of the enzymatic preparation. In this connection, Dashek and Micales (45) have discussed the factors that must be considered when assaying enzyme activity. In addition, they review protein quantification. 

For reproducible expression analysis and protein quantification MS methods based on isotopic labeling are available. They were designed in conjunction with two or more dimensional chromatographic peptide separation coupled online to MS and require advanced bioinformatics input to analyze the complex data sets in a reasonable time frame. This is also true for the alternative fluorescence-based technology of differential gel electrophoresis (DIGE Fig. 10.6) with tailor-made software which allows statistical validation of multiple data sets. 

Valanne A, Suojanen J, Peltonen J, Soukka T, Hanninen P, Harma H (2009) Multiple sized europium(III) chelate-dyed polystyrene particles as donors in FRET - an application for sensitive protein quantification utilizing competitive adsorption. Analyst 134 980-986... 

The range of the CE-SDS method depends on each individual protein molecule. Generally the method is linear from 0.5 to 2.0 mg/mL for both UV/PDA and laser-induced fluorescent (LIE) methods.Although protein concentrations as high as 3.0 mg/mL can be used for protein quantification, considering the SDS to protein ratio required, high protein concentrations are not recommended. LOD/LOQ values are different for UV/PDA versus LIE detection. For LIE detection with 5-carboxytetramethylrhodamine succinimidyl... 

Brase JC, Mannsperger H, Sultmann H et al (2011) Antibody-mediated signal amplification for reverse phase protein array-based protein quantification. Methods Mol Biol 785 55-64... 

Edman-based protein sequencing, immunostaining, SDS-capillary gel electrophoresis for protein quantification at picomolar range... 

Protein quantification is an important step for handling protein samples for isolation and characterization, and is a prerequisite step before submitting proteins for chromatographic, electrophoretic, or immunochemical analysis and separation. The methods included in this unit are colorimetric measurements, whose procedures are faster, simpler, and less laborious than those based on estimation of total nitrogen content (vnitbi.2). 

Hawkins, B.K. and Honigs, D.E. 1987. A comparison of spectroscopic techniques for protein quantification in aqueous solutions. Am. Biotechnol. Lab. 5 26-37. 

The peptide/protein quantification methods used in these assays are somewhat imprecise, because the color yield per mole or per mi lligram peptide is not consistent between peptides or proteins of different composition (Davis, 1988). Amounts of product are thus typically reported in terms of BSA equivalents, because calibration curves are typically prepared with this protein, and, again, absolute mole or absolute mass values are not readily obtained. This same limitation applies to the azosubstrates, since the color yield per mass peptide will be dependent on that given peptide s extent of derivitization. 

N.J. Kruger, The Bradford method for protein quantification. In J.M. Walker (Ed.), The Protein Protocols Handbook, Humana Press, New Jersey, 2002, pp. 15-21. 

Protein quantification in a solution is an extremely important problem no specific activity can be reported without employing both an activity assay and a protein quantification assay. The outcome of a protein quantification assay is a measure of total protein in a sample solution, routinely reported in mg mlT1 or mg L-1. Compared to an ideal assay, which should be accurate, very sensitive, reliable, fast, and easy to perform, all commonly employed assays for total protein suffer several shortcomings. 

Zhang HT, Kacharmina JE, Miyashiro K, Greene MI, Eberwine J. Protein quantification from complex protein mixtures using a proteomics methodology with single-cell resolution. Proc Natl Acad Sri USA 2001 98(10) 5497-5502. 

LABELING AND LABEL-FREE APPROACHES FOR PROTEIN QUANTIFICATION... 

The interest of the consortium in developing and testing new tools for proteome analysis was directed to solutions for particular technical problems concerning sample preparation, the 2D PAGE system, protein quantification, and the development of UniClone sets (nonredundant cDNA expression library) from the adult human brain to be used for creating clinically relevant biochips. These... 

Table 3.1 The most commonly used protein staining methods for proteome analysis using 2-DE. Besides the low detection limit of the applied staining methods, the linear dynamic range for protein quantification is another important parameter for a global description of a proteome.

Ross, P. L., Huang, Y. N., Marchese, ). N., Williamson, B., Parker, K., Hattan, S., Khainovski, N., Piliai, S., Dey, S., Daniels, S., etal. (2004). Multiplexed protein quantification in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol. Cell Proteomics 3, 1154-1169. 

Biomedical analysis analysis of DNA, proteins, quantification of P, Zn, Cu, and Fe levels in Alzheimer s and Parkinson s diseases, toxicity of elements [88, 89]... 

Purification of antisera (10 ml) for mass spectrometric epitope analysis was per-formedon Protein A-Sepharose (Amersham Biosciences) according to manufacturer s instructions. Protein quantification was performed using the micro-BCA kit (Pierce, Rockford) positive elution fractions were concentrated to 7 (ig/ml. 

To demonstrate MT-MEC as a useful platform for protein quantification, a simple surface biotin-avidin assay was constructed[15,16]. In the assay, biotinylated-BSA is incubated on both silvered and glass substrates (Figure 15.5). HRP-streptavidin is then added to the surface, locaiizing the enzyme catalyst in close proximity to the silver for MT-MEC. The peroxide and Acridan (iumophore) are then added to initiate the chemiluminescence reaction. While this assay in essence determines BSA concentration, this model assay could indeed be fashioned to both iocaiize and sense other proteins / DNAs of interest. 

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