A Path to Protein Quantitation: PBMCs to Western Blot


  • Sufaya Jameel Department of Zoology, School of Sciences, Maulana Azad National Urdu University, Hyderabad, Telangana https://orcid.org/0000-0003-1911-2857
  • Parveen Jahan Department of Zoology, School of Sciences, Maulana Azad National Urdu University, Hyderabad, Telangana https://orcid.org/0000-0003-3203-1816
  • Subbiah Ramasamy Department of Biochemistry, School of Biological Science, Madurai Kamaraj University, Madurai-625021, Tamil Nadu




Proteins are biopolymers made up of one or more long chains of amino acid residues. These proteins mediate thousands of metabolic pathways that determine how an organism functions. When proteins are thoroughly investigated, they can provide information about health and disease. A variety of methods have been developed to quantify both complex protein mixtures for total protein content as well as a single type of protein. This manuscript covers the methodologies standardized in our lab based on previously published protocols, beginning with the isolation of peripheral blood mononuclear cells (PBMCs) from human blood samples, cell viability testing, cell counting, total protein extraction methods, and protein quantification and estimation techniques, which enable a researcher to find the protocol from PBMCs to Western blot in a single manuscript.


PBMCs, Western blot, Chemiluminescence


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M. J. Lopez and S. S. Mohiuddin, “Biochemistry, Essential Amino Acids,” StatPearls, Mar. 2022.

T. Sanvictores and F. Farci, “Biochemistry, Primary Protein Structure,” StatPearls, pp. 1–9, 2020.

N. Boodhun, “Protein analysis: key to the future,” BioTechniques, vol. 64, no. 5, pp. 197–201, May 2018, doi: 10.2144/BTN-2018-0055.

C. H. Lee, “A Simple Outline of Methods for Protein Isolation and Purification,” Endocrinology and Metabolism, vol. 32, no. 1, p. 18, Mar. 2017, doi: 10.3803/ENM.2017.32.1.18.

K. Kallberg, H. O. Johansson, and L. Bulow, “Multimodal chromatography: An efficient tool in downstream processing of proteins,” Biotechnol J, vol. 7, no. 12, pp. 1485–1495, Dec. 2012, doi: 10.1002/BIOT.201200074.

S. S. Nielsen, Food Analysis, 5th ed., . 5th ed., in Food Science Text Series. Springer Cham, 2017. doi: 10.1007/978-3-319-45776-5.

A. Puleo, C. Carroll, H. Maecker, and R. Gupta, “Isolation of Peripheral Blood Mononuclear Cells Using Vacutainer® Cellular Preparation Tubes (CPTTM),” Bio Protoc, vol. 7, no. 2, 2017, doi: 10.21769/BIOPROTOC.2103.

Y. Grigoryev, “Cell Counting With a Hemocytometer: As Easy as 1, 2, 3...,” Bite Size Bio, Jun. 29, 2021. https://bitesizebio.com/13687/cell-counting-with-a-hemocytometer-easy-as-1-2-3/ (accessed Mar. 28, 2023).

L. L. Y. Chan, W. L. Rice, and J. Qiu, “Observation and quantification of the morphological effect of trypan blue rupturing dead or dying cells,” PLoS One, vol. 15, no. 1, p. e0227950, Jan. 2020, doi: 10.1371/JOURNAL.PONE.0227950.

M. Betti, C. Ciacci, S. Abramovich, and F. Frontalini, “Protein Extractions from Amphistegina lobifera: Protocol Development and Optimization,” Life (Basel), vol. 11, no. 5, May 2021, doi: 10.3390/LIFE11050418.

L. C. M. Ngoka, “Sample prep for proteomics of breast cancer: proteomics and gene ontology reveal dramatic differences in protein solubilization preferences of radioimmunoprecipitation assay and urea lysis buffers,” Proteome Sci, vol. 6, Oct. 2008, doi: 10.1186/1477-5956-6-30.

M. Johnson, “Protein Quantitation,” Materials and Methods, vol. 2, Feb. 2012, doi: 10.13070/MM.EN.2.115.

F. He, “Laemmli SDS PAGE,” Bio Protoc, vol. 1, no. 11, pp. 1–4, Jun. 2011, Accessed: Mar. 28, 2023. [Online]. Available: https://bio-protocol.org/pdf/Bio-protocol80.pdf

M. M. Bradford, “A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding,” Anal Biochem, vol. 72, no. 1–2, pp. 248–254, May 1976, doi: 10.1016/0003-2697(76)90527-3.

C. M. Stoscheck, “[6] Quantitation of protein,” Methods Enzymol, vol. 182, no. C, pp. 50–68, Jan. 1990, doi: 10.1016/0076-6879(90)82008-P.

S. C. Silvério, S. Moreira, A. M. F. Milagres, E. A. Macedo, J. A. Teixeira, and S. I. Mussatto, “Interference of some aqueous two-phase system phase-forming components in protein determination by the Bradford method,” Anal Biochem, vol. 421, no. 2, pp. 719–724, Feb. 2012, doi: 10.1016/J.AB.2011.12.020.

A. B. Nowakowski, W. J. Wobig, and D. H. Petering, “Native SDS-PAGE: High Resolution Electrophoretic Separation of Proteins With Retention of Native Properties Including Bound Metal Ions,” Metallomics, vol. 6, no. 5, p. 1068, 2014, doi: 10.1039/C4MT00033A.

“SDS-PAGE Protocol | Rockland.” https://www.rockland.com/resources/sds-page-protocol/ (accessed Mar. 29, 2023).

T. Mahmood and P.-C. Yang, “Western blot: technique, theory, and trouble shooting,” N Am J Med Sci, vol. 4, no. 9, pp. 429–434, Sep. 2012, doi: 10.4103/1947-2714.100998.

A. K., “Improving quantification accuracy for western blots,” Image Analysis, Sep. 2006.

A. Lal, S. R. Haynes, and M. Gorospe, “Clean western blot signals from immunoprecipitated samples,” Mol Cell Probes, vol. 19, no. 6, p. 385, Dec. 2005, doi: 10.1016/J.MCP.2005.06.007.

J. Pourahmad and A. Salimi, “Isolated Human Peripheral Blood Mononuclear Cell (PBMC), a Cost Effective Tool for Predicting Immunosuppressive Effects of Drugs and Xenobiotics,” Iranian Journal of Pharmaceutical Research, vol. 14, no. 4, p. 979, 2015, Accessed: Mar. 29, 2023. [Online]. Available: https://pubmed.ncbi.nlm.nih.gov/26664364/

S. P, “PBMCs: Applications in Research,” News Medical Life Sciences. https://www.news-medical.net/life-sciences/PBMCs-Applications-in-Research.aspx (accessed Mar. 29, 2023).

T. L. Riss et al., “Cell Viability Assays,” Assay Guidance Manual, Jul. 2016. https://www.ncbi.nlm.nih.gov/books/NBK144065/

K. Gavini and K. Parameshwaran, “Western Blot,” StatPearls, Apr. 2022. https://www.ncbi.nlm.nih.gov/books/NBK542290/

M. R. White and E. D. Garcin, “The sweet side of RNA regulation: glyceraldehyde-3-phosphate dehydrogenase as a noncanonical RNA-binding protein,” Wiley Interdiscip Rev RNA, vol. 7, no. 1, p. 53, Jan. 2016, doi: 10.1002/WRNA.1315.




How to Cite

S. Jameel, P. Jahan, and S. Ramasamy, “A Path to Protein Quantitation: PBMCs to Western Blot”, Int. J. Methodol., vol. 2, no. 1, pp. 12–27, Apr. 2023.

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