Protein measurement

Lowry, O.H. Rosebrough, N.J. Farr, A.L. and Randall, R.J. Protein measurement with folin phenol reagent. J Biol Chem 193 265-275, 1951. Munson, P.J.. and Rodbard, D. LIGAND A versatile computerized approach for characterization of ligand binding systems. Anal Biochem 107 220-237. 1980. 

Preejith P.V., Lim C.S., Kishen A., John M.S., A. Asundi Total protein measurement using a fiber-optic evanescent wave-based biosensor, Biotechn.Lett. 2003 25 105-110. 

Perhaps a more serious challenge to the scalability of the BioCD is the limit of understanding of what multiple measurements mean biologically. Today, when a doctor orders a panel of clinical tests, perhaps only a dozen or so protein measurements are made. The reason for this small number, in the face of 10,000 available blood proteins, is that doctors would not know what to do with all the measurements that could be made available. Therefore, the prospect of making thousands of analyte measurements per patient today is largely meaningless. 

Protein measurement with the Folin phenol reagent. J. ... 

Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. Protein measurement with Folin phenol reagent. J. Biol. 

If analytical methods are at the heart of biopharmaceutical development and manufacturing, then protein concentration methods are the workhorse assays. A time and motion study of the discovery, development, and manufacture of a protein-based product would probably confirm the most frequently performed assay to be protein concentration. In the 1940s Oliver H. Lowry developed the Lowry method while attempting to detect miniscule amounts of substances in blood. In 1951 his method was published in the Journal of Biological Chemistry. In 1996 the Institute for Scientific Information (ISI) reported that this article had been cited almost a quarter of a million times, making it the most cited research article in history. This statistic reveals the ubiquity of protein measurement assays and the resilience of an assay developed over 60 years ago. The Lowry method remains one of the most popular colorimetric protein assays in biopharmaceutical development, although many alternative assays now exist. 

Delayed uptake of bilirubin has been described in mutant Southdown sheep (CIO), suggesting a deficiency in cytoplasmic organic anion-binding protein. Measurements of the conjugating enzymes are desirable. Conceivably, a decreased conjugating capacity could influence the uptake, as is suggested from studies on patients with Gilbert s syndrome (B14, B15,B18). 

The independent determination of molecular masses by SDS-PAGE is impossible. To estimate the molecular mass of a protein, measure the path of that protein or calculate its Rf value (distance of the protein from origin/distance of electrophoresis front from origin) and compare these values with that of marker proteins, i.e., proteins with independently determined molecular masses. This method is successful only if the protein of interest behaves regularly in SDS-PAGE, i.e., it is totally unfolded by SDS, has a rod-like shape, and the SDS/protein ratio is the same for the unknown and the marker protein. Especially highly hydrophobic proteins and glycoproteins often deviate from these assumptions. 

Expect follow-up tests, such as ESR, C-reactive protein measurement, and urinalysis... 

Kingsmore, S.F. (2006) Multiplexed protein measurement technologies and applications of protein and antibody arrays. Nett Rev Drug Discov. 5, 310-20. 

Perhaps if you have a resourceful and supportive national measurement institute, you might be able to persuade it to organize an interlaboratory certification to provide appropriate calibration materials, as has been done in Australia by the grain industry for protein measurements. 

Lowry OH, Roseborough NJ, Farr AL, Randall RJ (1951) Protein measurements with Folin phenol reagent. J Biol Chem 193 265-275... 

Brown, A.J., Jarvis, K., and Hyland, K. 1989. Protein measurement using bicinchoninic acid Elimination of interfering substances. Anal. Biochem. 180 136-139. 

Because stray light can affect the linearity of absorbance versus concentration, absorbance values >2.0 should not be used for sample proteins measured by the A28o or A2()<, method. Samples with absorbance >2.0 should be diluted further in the appropriate buffer to obtain absorbances <2.0. 

Measurement of total protein is useful, as it indicates the degree of purification at each step. However, unless the next step critically depends on how much protein is present, total protein measurement is not extremely important a small sample can be put aside and measured later, when the purification is complete. It is, however, very important to know how much protein is present in the final, presumed pure sample, as this will indicate the specific activity (if the protein has an activity), which can be compared with other preparations. The general object is to obtain as high a specific activity as possible (taking into account recovery considerations), which means retaining as much of the desired protein as possible while ending up with as little total protein as possible. 

Structure of specific sites this includes structures of small molecules whether they be carbohydrates, lipids, small proteins or complexes between segments of DNA and proteins Measurement of concentrations or rate of reactions Type and extent of secondary structure... 

To determine the F/P ratio of the labeled protein, measure the absorbance of the purified preparation at 345 and 280 nm. Ratios of 345 /280 nm within the range 0.3 to 0.8 usually result in fluorescent conjugates with a good balance of high-intensity luminescence, low nonspecific binding, and excellent retention of biological activity within the protein component. 

Figure 3. LMW endoglucanase from Sephadex G-75 column chromatography. (—) Protein (measured at A280nm), (-X-) reducing sugar...

In brief, determining the structure of a protein by X-ray crystallography entails growing high-quality crystals of the purified protein, measuring the directions and intensities of X-ray beams diffracted from the crystals, and using a computer to simulate the effects of an objective lens and thus produce an... 

Confluent cells were incubated with a wide variety of radioactive isotopes for 48 hrs in medium containing either FCS or LPDS. Subsequently, the medium was removed and the monolayer washed twice with ice cold PBS-2% BSA and five times with ice cold PBS. The entire monolayer was solubilized in 1 n NaOH and suitable aliquots used for radioactivity and protein measurements. 

Pollack, L., Tate, M. W., Damton, N. C., Knight, J. B., Gruner, S. M., Eaton, W. A., and Austin, R. H. (1999). Compactness of the denatured state of a fast-folding protein measured by submillisecond small-angle X-ray scattering. Proc. Natl. Acad. Sci. USA 96, 10115-10117. 

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