Protein estimation

Protein determination is necessary to estimate the amount of protein in the sample, to normalise against the protein concentration or during purification procedures. Depending on the amount of sample, accuracy and presence of interfering agents, one needs to decide on the method to be used. For accurate quantification, the sample protein is compared with a known amount of a standard protein which could either be the commonly used bovine serum albumin (BSA) or it could sometimes be immunoglobulin G (IgG). The various methods and their specifications are outlined below  

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The greatest surprise provided by the results to date has been the apparently low number of genes encoding proteins, estimated to lie between 30,000 and 40,000. The higher number could increase as new data are obtained. This number is approximately twice that found in the roundworm (19,099) and three times that of the fruit... 

Haemophilus influenzae type b Cultures of H. influenzae type b 1 Separation of capsular polysaccharide 2 Conjugation with a protein Estimation of capsular polysaccharide content ... 

Protein estimation Proteins were determined by the method of Lx)wry et al. (1951), using bovine serum albumin as standard. 

Fig. B.l Standard graph for protein estimation using known concentration of BSA as standard.

It is very often observed that during a purification process the differences increase between the real amounts of a protein and the values obtained by any method, e.g., total enzyme activity, because the measured signal produced by a protein mixture differs from that of a pure protein. Furthermore, the amount of a given protein determined by a distinct protocol differs from the expected amount by portioning, as shown in Table 1.1. To avoid additional mistakes with the already uncertain process, the protein estimation method should not be changed during a purification process. 

The Kjeldahl total nitrogen determination method is not very sensitive, hut it suits well for analyzing insoluble samples without preceding disintegration. Automated Kjeldahl protein estimations are used especially in food analysis. 

Add 0.5 -1.0 ml of Soln. G to the precipitate and heat the mixture to 90 °C for 10 min. After cooling, the samples are centrifuged as described above and the supernatant is used for protein estimation by the Lowry protocol (Protocol 1.1.1.1) third supernatant. 

EXAMPLE 4.4 Extent of Hydration of a Protein Molecule from Intrinsic Viscosity Measurements. Suppose an aqueous solution of a spherical protein molecule shows an intrinsic viscosity of 3.36 cm3 g 1. Taking p2 = 1.34 g cm 3 for the dry protein, estimate the extent of hydration of the protein. 

Students will isolate intact mitochondria from beef heart and fractionate them to prepare submitochondrial particles. Each fraction will be characterized by protein estimation by the biuret method and measurement of malate dehydrogenase and monoamine oxidase activity. 

Plus Protein Assay Reagent was used, the total protein (IgG) concentration in the sample would be underestimated by -40%. (From Table B1.1. 5, the response ratio for IgG is -0.58 for IgG compared to 1.00 for BSA.) If the BCA Protein Assay Reagent was used, the total protein (IgG) concentration in the sample would be overestimated by -15%. (From Table B1.1.5, the response ratio for IgG is -1.15 for IgG compared to 1.00 for BSA.) On the other hand, if BGG had been used for both standard curves, the total protein estimates for the sample would have been in much greater agreement between the two methods. 

Jaceldo-Siegl K, Fraser GE, Chan J, Franke AA, Sabate J. 2008. Validation of soy protein estimates from a food-frequency questionnaire with repeated 24-h recalls and isoflavonoid excretion in overnight urine in a western population with a wide range of soy intakes. Am J Clin Nutr 87 1422-1427. 

Crude protein and total lysine plus furosine Total nitrogen was determined by macro Kjeldahl digestion and protein estimated as N x 6.25. Total lysine values were obtained from conventional amino acid analyses carried out on 500-mg samples following digestion with 800 ml of 6M HC1 under reflux by use of a Biotronic LC 6000 or Kontron Liquimat III amino acid analyzer. Furosine was determined in the same way using 300 ml 7.8 M HC1 as described in ( 6 ) with an amino acid analyzer ( 7 ). 

Take 100 /xl samples for protein estimation and add 3 ml reagent. Mix well and leave for 4 min. Read A595 within one hour. A standard curve can be made using... 

The Degree, of Folding of Native Proteins Estimated from the Change in Optical Rotation... 

E. coli is a rod-shaped bacterium about 2 long and 1 /a in diameter. The average density of a cell is 1.28 g/ml. Approximately 13.5% of the wet weight of E. coli is soluble protein. Estimate the number of molecules of a particular enzyme per cell if the enzyme has a MW of 100,000 and represents 0.1% of the total soluble protein. 

Fig. 1. Protein standard curve. Duplicate or triplicate sample readings are averaged and concentration is extrapolated from the standard curve. Standard deviation of protein estimations should be <5%.

Figure 1.5. Relationship between actual and measured BSA concentration in six samples, showing that this method produces comparable results to the Bradford and BCA total protein assays. [Reprinted, with permission, from K. C. Bible, S. A. Boemer, and S. H. Kaufmann, Anal. Biochem. 267, 1999, 217-221. A One-Step Method for Protein Estimation in Biological Samples Nitration of Tyrosine in Nitric Acid. Copyright 1999 by Academic Press.]...

Figure 24-5 Frequency distribution of quantitative results combined with returns of "nil, zero, or not detected for distributions of salt solution and normal urine. Quality control of total protein measurement demonstrates the widespread variation in results achieved using different methods, (from Chombers RE, Bullock DG, Wh/cfier JT. Urinary total protein estimation Fact or fiction Nephron 1989 53 33. Reproduced with permission of S. Karger AG, Basel, Switzerland.)...

Chambers RE, Bullock DG, Whicher JT. Urinary total protein estimation Fact or fiction Nephron 1989 53 33-6. 

Chambers RE, Bullock DG, Wliicher JT. External quality assessment of total urinary protein estimation in the United Kingdom. Ann Clin Biochem 1991 28 467-73. 

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