Bradford assay as a high-throughput bioanalytical screening method for conforming pathophysiological state of the animal

  • Abhishesh Kumar Mehata Department of Pharmaceutical Engineering and Technology, Indian Institute of Technology (BHU), Varanasi–221005, India
  • Deepa Dehari Department of Pharmaceutical Engineering and Technology, Indian Institute of Technology (BHU), Varanasi–221005, India

Abstract

Proteins are the essential components of the tissues that play a key role in the body. Its expression in the cell or tissue under a specified set of conditions and at a particular time regulates the different body conditions either as a normal body function or as a disease state. Protein is an important building block of muscles, skin, cartilage, bones and blood. Bradford assay is a reliable advanced and cost-effective protein estimation test for determining the exact concentration of protein in different tissues of the animal. In this study, we have taken a rat suffering from protein deficiency disorder and total protein concentration in the heart, brain, liver, blood and kidney was determined. It was found that the total protein concentration in different tissues of rat i.e., heart, brain, liver, plasma and kidney was found to be 8.39 ± 0.75, 10.46 ± 0.76, 6.74 ± 0.39, 8.12 ± 0.32 mg/g of tissue and 61.27 ± 0.95 mg/mL of plasma respectively (mean ± SEM). As compared to earlier published reports the total protein concentration in different tissues like hear, brain, liver and kidney found much lower to standard value as reported by Beyer, the reason behind obtaining this kind of results may be due to the presence of insufficient amount of the protein content in different tissue of animal as suffering from protein degeneration disorder. The rat was unable to digest and store the protein or catabolism was much faster than


anabolism.


Keywords: Anabolism, Bradford assay, Catabolism, Protein estimation.

Keywords: Anabolism, AnabolismAnabolism, Protein estimation

Downloads

Download data is not yet available.

References

1. Graslund S, Nordlund P, Weigelt J, Hallberg BM, Bray J, Gileadi O, Knapp S, Oppermann U, Arrowsmith C, Hui R, Ming J. Protein production and purification. Nature methods, 2008; (2):135.
2. Bradley BP, Kalampanayil B, O’Neill MC. Protein expression profiling. In Two-Dimensional Electrophoresis Protocol. Springer Humana Press,.2009; 455-468.
3. Maehre HK, Dalheim L, Edvinsen GK, Elvevoll EO, Jensen IJ. Protein determination—method matters. Foods, 2018; 7(1):5.
4. Zheng K, Wu L, He Z, Yang B, Yang Y. Measurement of the total protein in serum by biuret method with uncertainty evaluation. Measurement, 2017; 1(112):16-21.
5. Mircean C, Shmulevich I, Cogdell D, Choi W, Jia Y, Tabus I, Hamilton SR, Zhang W. Robust estimation of protein expression ratios with lysate microarray technology. Bioinformatics, 2005; 21(9):1935-42.
6. Kirazov LP, Venkov LG, Kirazov EP. Comparison of the Lowry and the Bradford protein assays as applied for protein estimation of membrane-containing fractions. Analytical biochemistry, 1993; 208(1):44-48.
7. Martina VR, Vojtech K. A comparison of Biuret, Lowry and Bradford methods for measuring the egg’s proteins. Proceedings of the Mendel Net, 2015:394-398.
8. Kruger NJ. The Bradford method for protein quantitation, p 15–21. The protein protocols handbook, 2nd ed. Humana Press, Totowa, NJ. 2002.
9. Shire SJ, Shahrokh Z, Liu JU. Challenges in the development of high protein concentration formulations. Journal of pharmaceutical sciences, 2004; 93(6):1390-402.
10. Zor T, Selinger Z. Linearization of the Bradford protein assay increases its sensitivity: theoretical and experimental studies. Analytical biochemistry, 1996; 236(2):302-308.
11. Ohnishi ST, Barr JK. A simplified method of quantitating protein using the biuret and phenol reagents. Analytical biochemistry, 1978; 86(1):193-200.
12. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. Journal of biological chemistry, 1951; (193):265-75.
13. Oosta GM, Mathewson NS, Catravas GN. Optimization of Folin-Ciocalteu reagent concentration in an automated Lowry protein assay. Analytical biochemistry, 1978; 89(1):31-34.
14. Compton SJ, Jones CG. Mechanism of dye response and interference in the Bradford protein assay. Analytical biochemistry, 1985; 151(2):369-374.
15. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical biochemistry, 1976; 2(1-2):248-254.
16. Brown RE, Jarvis KL, Hyland KJ. Protein measurement using bicinchoninic acid: elimination of interfering substances. Analytical biochemistry, 1989; 180(1):136-139.
17. Smith PE, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano M, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC. Measurement of protein using bicinchoninic acid. Analytical biochemistry, 1985; 150(1):76-85.
18. Noble JE, Bailey MJ. Quantitation of protein. In Methods in enzymology, 2009; 463:73-95.
19. Sapan CV, Lundblad RL, Price NC. Colorimetric protein assay techniques. Biotechnology and applied Biochemistry, 1999; (2):99-108.
20. Der-Balian GP, Gomez B, Masino RS, Parce JW. A fluorometric method for determining the degree of biotinylation of proteins. Journal of immunological methods, 1990; 126(2):281-285.
21. Iwata J, Nishikaze O. New micro-turbidimetric method for determination of protein in cerebrospinal fluid and urine. Clinical Chemistry, 1979; 25(7):1317-1319.
22. Nurahmanto D. Development and validation of UV spectrophotometric method for quantitative estimation of Promethazine HCl in phosphate buffer saline pH 7.4. International Current Pharmaceutical Journal, 2013; 2(8):141-142.
23. Sedlak J, Lindsay RH. Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent. Analytical biochemistry. 1968; 25:192-205.
24. Kruger NJ. The Bradford method for protein quantitation. In The protein protocols handbook, 2009; 17-24.
25. Beyer RE. A rapid biuret assay for protein of whole fatty tissues. Analytical biochemistry, 1983; 129(2):483-485.
26. Kocic G, Veljkovic A, Kocic H, Colic M, Mihajlovic D, Tomovic K, Stojanovic S, Smelcerovic A. Depurinized milk downregulates rat thymus MyD88/Akt/p38 function, NF-κB-mediated inflammation, caspase-1 activity but not the endonuclease pathway: In vitro/in vivo study. Scientific reports. 2017; 7:41971.
27. Oseas da Silva MA, Arruda MA. Mechanization of the Bradford reaction for the spectrophotometric determination of total proteins. Analytical biochemistry, 2006; 351(1):155-157.
28. Eaton SL, Roche SL, Hurtado ML, Oldknow KJ, Farquharson C, Gillingwater TH, Wishart TM. Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting. PloS one, 2013; 8(8):72457.
29. Welinder C, Ekblad L. Coomassie staining as loading control in Western blot analysis. Journal of proteome research, 2011; 10(3):1416-1419.
30. Verrills NM. Clinical proteomics: present and future prospects. Clinical Biochemist Reviews, 2006; 27(2):99.
Statistics
8 Views | 11 Downloads
How to Cite
1.
Mehata A, Dehari D. Bradford assay as a high-throughput bioanalytical screening method for conforming pathophysiological state of the animal. JDDT [Internet]. 15Feb.2020 [cited 27Feb.2020];10(1-s):105-10. Available from: http://jddtonline.info/index.php/jddt/article/view/3921