Moleküler Analiz Yöntemleri: PCR, RT-qPCR, ELISA, Western Blot ve Protein Düzeyi Ölçümü
Özet
Moleküler ve immünokimyasal analiz yöntemleri, biyolojik sistemlerde genetik bilginin ifade edilmesi ve bunun protein düzeyindeki fonksiyonel karşılığının ortaya konulmasında temel bir rol üstlenmektedir. Bu bölümde, biyomedikal araştırmalarda yaygın olarak kullanılan ELISA, PCR, RT-qPCR, Western Blot ve protein düzeyi ölçüm yöntemleri; temel çalışma prensipleri, uygulama alanları ve analitik güçlü yönleriyle birlikte bütüncül bir çerçevede ele alınmaktadır. ELISA, özgül antijen-antikor etkileşimlerine dayalı yapısı sayesinde sitokinler, hormonlar, büyüme faktörleri ve antikorlar gibi çok sayıda biyomolekülün kantitatif veya yarı-kantitatif ölçümüne olanak tanır ve klinik ile preklinik çalışmalar arasında önemli bir köprü oluşturur. PCR ve RT-qPCR teknikleri, özgül DNA dizilerinin çoğaltılması ve RNA moleküllerinden gen ekspresyonunun gerçek zamanlı olarak kantitatif değerlendirilmesi yoluyla, hücresel düzeydeki transkripsiyonel değişimlerin yüksek duyarlılıkla analiz edilmesini sağlar. Floresan tabanlı tespit sistemleri, Ct değerine dayalı kantifikasyon yaklaşımları ve uygun referans genlerle yapılan normalizasyon stratejileri, RT-qPCR verilerinin doğruluğu ve biyolojik anlamlılığı açısından kritik öneme sahiptir. Protein analizinde temel yöntemlerden biri olan Western Blot, proteinlerin moleküler ağırlıklarına göre ayrılması ve özgül antikorlar aracılığıyla tespit edilmesi sayesinde protein ekspresyon düzeyleri, izoformlar ve post-translasyonel modifikasyonlar hakkında ayrıntılı bilgi sunar. Bununla birlikte Bradford, Lowry ve BCA gibi toplam protein kantifikasyon yöntemleri, örneklerin standardizasyonu ve deneysel karşılaştırmalar için vazgeçilmezdir. Bu yöntemlerin birlikte ve tamamlayıcı şekilde kullanılması, gen ekspresyonu ile protein düzeyi arasındaki ilişkinin bütüncül olarak değerlendirilmesine olanak tanır. Güvenilir ve tekrarlanabilir sonuçlar elde edebilmek için uygun örnek hazırlığı, deneysel kontroller, analitik validasyon adımları ve istatistiksel değerlendirmelerin titizlikle planlanması gerekmektedir. Sonuç olarak, bu bölümde ele alınan analiz yaklaşımları, biyolojik süreçlerin moleküler temellerinin aydınlatılması ve hastalık mekanizmalarının anlaşılmasında güçlü ve vazgeçilmez araçlar sunmaktadır.
Molecular and immunochemical analysis methods play a fundamental role in elucidating the expression of genetic information and its functional counterparts at the protein level in biological systems. This section presents a comprehensive overview of ELISA, PCR, RT-qPCR, Western blot, and protein quantification methods that are widely used in biomedical research, addressing their basic principles of operation, application areas, and analytical strengths within an integrated framework. ELISA enables the quantitative or semi-quantitative measurement of a wide range of biomolecules, including cytokines, hormones, growth factors, and antibodies, through its structure based on specific antigen–antibody interactions, thereby serving as an important bridge between clinical and preclinical studies. PCR and RT-qPCR techniques allow highly sensitive analysis of transcriptional changes at the cellular level by enabling the amplification of specific DNA sequences and the real-time quantitative evaluation of gene expression from RNA molecules. Fluorescence-based detection systems, Ct value–based quantification approaches, and normalization strategies employing appropriate reference genes are critical for ensuring the accuracy and biological relevance of RT-qPCR data. Western blot, one of the fundamental methods in protein analysis, provides detailed information on protein expression levels, isoforms, and post-translational modifications by separating proteins according to their molecular weight and detecting them through specific antibodies. In addition, total protein quantification methods such as the Bradford, Lowry, and BCA assays are indispensable for sample standardization and comparative experimental analyses. The combined and complementary use of these methods allows a holistic evaluation of the relationship between gene expression and protein levels. To obtain reliable and reproducible results, careful planning of sample preparation, experimental controls, analytical validation steps, and statistical evaluations is essential. In conclusion, the analytical approaches discussed in this section represent powerful and indispensable tools for elucidating the molecular foundations of biological processes and for understanding disease mechanisms.
Referanslar
Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009;55(4):611–622
Li Z, Yang X, Yang B, Yang J, Tao C, Zhang D, et al. A comprehensive review of methodological and technological advancement in PCR during the last 15 years. Biotechnol Adv. 2025;85:108719
Shafeeq NK. Polymer chain reaction (PCR): Principle and applications. Ibn Al-Haitham J Pure Appl Sci. 2021;34(4):35–44
Green M, Sambrook J. Nested polymerase chain reaction (PCR). Cold Spring Harb Protoc. 2019;2019(2):pdb.prot095182.
Kang BH, Lee Y, Yu ES, Na H, Kang M, Huh HJ, et al. Ultrafast and real-time nanoplasmonic on-chip polymerase chain reaction for rapid and quantitative molecular diagnostics. ACS Nano. 2021;15(6):10194–10202
Mullis KB, Faloona FA. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 1987;155:335–350.
Innis MA, Gelfand DH, Sninsky JJ, White TJ, editors. PCR protocols: A guide to methods and applications. San Diego: Academic Press; 1990
Botstein D, White RL, Skolnick M, Davis RW. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am J Hum Genet. 1980;32(3):314–331
Higuchi R, von Beroldingen CH, Sensabaugh GF, Erlich HA. DNA typing from single hairs. Nature. 1988;332(6164):543–546
Corman VM, Landt O, Kaiser M, et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill. 2020;25(3):2000045
Praité A, Lambert J, Delpy L, Hayek S. Guidelines for RNA analysis by reverse transcription quantitative polymerase chain reaction. Methods Mol Biol. 2025;2962:3–13
Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res. 1996;6(10):986–994.
Kubista M, Andrade JM, Bengtsson M, Forootan A, Jonák J, Lind K, et al. The real-time polymerase chain reaction. Mol Aspects Med. 2006;27(2–3):95–125
Bong D, Sohn J, Lee SV. Brief guide to RT-qPCR. Mol Cells. 2024;47(12):100141.
Bustin SA. RT-qPCR testing and performance metrics in the COVID-19 era. Int J Mol Sci. 2024;25(17):9326
Tao Y, Yue Y, Qiu G, Ji Z, Spillman M, Gai Z, et al. Comparison of analytical sensitivity and efficiency for SARS-CoV-2 primer sets by TaqMan-based and SYBR Green-based RT-qPCR. Appl Microbiol Biotechnol. 2022;106(5–6):2207–2218
Ramezani A. CtNorm: Real time PCR cycle of threshold (Ct) normalization algorithm. J Microbiol Methods. 2021;106267
Svingen T, Letting H, Hadrup N, Hass U, Vinggaard A. Selection of reference genes for quantitative RT-PCR (RT-qPCR) analysis of rat tissues under physiological and toxicological conditions. PeerJ. 2015;3.
Taylor S, Wakem M, Dijkman G, Alsarraj M, Nguyen M. A practical approach to RT-qPCR—Publishing data that conform to the MIQE guidelines. Methods. 2010;50(4):S1–S5.
Dieffenbach CW, Lowe TM, Dveksler GS. General concepts for PCR primer design. PCR Methods Appl. 1993;3(3):S30–S37.
Monfort-Lanzas P, Rusu EC, Parrakova L, et al. ExonSurfer: a web-tool to design primers at exon–exon junctions. BMC Genomics. 2024;25:594.
Svec D, Tichopád A, Novosadova V, Pfaffl M, Kubista M. How good is a PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency assessments. Biomol Detect Quantif. 2015;3:9–16.
Sahoo S, Mandal S, Das P, Bhattacharya S, Chandy M. An analysis of the standard curve parameters of cytomegalovirus, BK virus and hepatitis B virus quantitative polymerase chain reaction from a clinical virology laboratory in eastern India. Indian J Med Microbiol. 2021.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402–408
Harshitha R, Arunraj D. Real-time quantitative PCR: A tool for absolute and relative quantification. Biochem Mol Biol Educ. 2021;49:800–812.
Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002;3(7):RESEARCH0034
Huggett J, Dheda K, Bustin S, Zumla A. Real-time RT-PCR normalisation; strategies and considerations. Genes Immun. 2005;6(4):279–284
Hellemans J, Vandesompele J. Selection of reliable reference genes for RT-qPCR analysis. Methods Mol Biol. 2014;1160:19–26
Schrader C, Schielke A, Ellerbroek L, Johne R. PCR inhibitors—occurrence, properties and removal. J Appl Microbiol. 2012;113(5):1014–1026
Ririe KM, Rasmussen RP, Wittwer CT. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem. 1997;245(2):154–160
Yuan JS, Reed A, Chen F, Stewart CN Jr. Statistical analysis of real-time PCR data. BMC Bioinformatics. 2006;7:85
Alhajj M, Zubair M, Farhana A. Enzyme-linked immunosorbent assay. In: StatPearls. StatPearls Publishing; 2025.
Chiswick EL, Duffy E, Japp B, Remick D. Detection and quantification of cytokines and other biomarkers. In: Methods in Molecular Biology. Vol. 844. Humana Press; 2012. p. 15–30.
Menzel A, Samouda H, Dohet F, Loap S, Ellulu MS, Bohn T. Common and novel markers for measuring inflammation and oxidative stress ex vivo in research and clinical practice: Which to use regarding disease outcomes? Antioxidants. 2021;10(3):414
Ryznar R, Wong C, Onat E, Towne F, LaPorta A, Payton M. Principal component analysis of salivary cytokines and hormones in the acute stress response. Front Psychiatry. 2022;13:957545.
Sakamoto S, Putalun W, Vimolmangkang S, et al. Enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of plant secondary metabolites. J Nat Med. 2018;72:32–42
AAA Biotech. (n.d.). Sandwich ELISA explained: Principle, protocols, and best practices. Retrieved December 17, 2025, from https://www.aaabiotech.com/blogs/sandwich-elisa-explained-principle-protocols-and-best-practices
National Center for Biotechnology Information. Competitive binding assay. In: Immunoassay Methods–Assay Guidance Manual. 2025. https://www.ncbi.nlm.nih.gov/books/NBK92434
Zhu L, He J, Cao X, et al. Development of a double-antibody sandwich ELISA for rapid detection of Bacillus cereus in food. Sci Rep. 2016;6:16092
Engvall E, Perlmann P. Enzyme-linked immunosorbent assay (ELISA): Quantitative assay of immunoglobulin G. Immunochemistry. 1971;8(9):871–874.
Wild DG, editor. The immunoassay handbook: Theory and applications of ligand binding, ELISA and related techniques. 4th ed. Elsevier; 2013
Andreasson U, Perret-Liaudet A, van Waalwijk van Doorn LJ, Blennow K, Chiasserini D, Engelborghs S, et al. A practical guide to immunoassay method validation. Front Neurol. 2015;6:179.
Florescu DN, Boldeanu M-V, Șerban R-E, Florescu LM, Serbanescu M-S, Ionescu M, et al. Correlation of the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α, inflammatory markers, and tumor markers with the diagnosis and prognosis of colorectal cancer. Life. 2023;13(12):2261
Tighe PJ, Ryder RR, Todd I, Fairclough LC. ELISA in the multiplex era: Potentials and pitfalls. Proteomics Clin Appl. 2015;9(3–4):406–422.
Jaschke P. Simulated sandwich enzyme-linked immunosorbent assay for a cost-effective investigation of natural and engineered cellular signaling pathways. Biochem Mol Biol Educ. 2019;48(1):34–41.
Dutt TS, Spencer JS, Karger BR, Fox A, Obregon-Henao A, Podell BK, Anderson GB, Henao-Tamayo M. ELISA-R: An R-based method for robust ELISA data analysis. Front Immunol. 2024;15:1427526.
Zhu B, Van R, Wang H, Kuang S, Jia Y, Leon E, et al. Detection of misfolded proteins in neurodegenerative disease models with a highly sensitive chemiluminescence probe. bioRxiv. 2025
Yang M, Zhang A, Chen M, Cao J. Advances in circulating biomarkers for neurodegenerative diseases, traumatic brain injuries, and central nervous system tumors. Ann Lab Med. 2025;45:381–390
Portilho A, Lima G, De Gaspari E. Enzyme-linked immunosorbent assay: An adaptable methodology to study SARS-CoV-2 humoral and cellular immune responses. J Clin Med. 2022;11:1503
Bolton J, Chaudhury S, Dutta S, Gregory S, Locke E, Pierson T, Bergmann-Leitner E. Comparison of ELISA with electro-chemiluminescence technology for the qualitative and quantitative assessment of serological responses to vaccination. Malar J. 2020;19:225
Garzón V, Salvador J, Marco M, G-Pinacho D, Bustos R. Development and ELISA characterization of antibodies against colistin, vancomycin, daptomycin, and meropenem: A therapeutic drug monitoring approach. Antibiotics. 2024;13:600
Zalba S, Contreras-Sandoval A, Martišová E, Debets R, Smerdou C, Garrido M. Quantification of pharmacokinetic profiles of PD-1/PD-L1 antibodies by validated ELISAs. Pharmaceutics. 2020;12:595
She Z. Application of enzyme-linked immunosorbent assay in cancer detection. Transact Mater Biotechnol Life Sci. 2024
Zhang D, Li WZ, Han H. Improved ELISA for tumor marker detection using electro-readout mode based on label-triggered degradation of methylene blue. Biosens Bioelectron. 2019;126:800–805
Souto PC, Santos MR, Orozco AMO, Bento LD, Ramírez López CJ, Girardi FM, et al. Enzyme linked immunosorbent assay (ELISA) development for equine serum amyloid A (SAA) determination using recombinant proteins. Methods Protoc. 2025;8(2):37.
Share E, Mastellar S, Suagee Bedore J, Eastridge M. Validation of a commercial ELISA kit for non invasive measurement of biologically relevant changes in equine cortisol concentrations. Animals. 2024;14:2831.
Ruy P, McDevitt A, O’Connor I, O’Dwyer K. Using ELISA to detect pathogen antibodies in wild mammal carcasses: A systematic literature review. Mamm Rev. 2024;55:e12379
Posyniak A, Żmudzki J, Niedzielska J. Evaluation of sample preparation for control of chloramphenicol residues in porcine tissues by enzyme linked immunosorbent assay and liquid chromatography. Anal Chim Acta. 2003;483:307–311.
Gonzalez R, Seurynck Servoss S, Crowley S, Brown M, Omenn G, Hayes D, Zangar R. Development and validation of sandwich ELISA microarrays with minimal assay interference. J Proteome Res. 2008;7(6):2406–2414
Riolo G, Biagini V, Guerrini N, Roscia G, Antonelli R, Giglioli G, et al. Design and validation of a semi quantitative microneutralization assay for human metapneumovirus A1 and B1 subtypes. Sci Rep. 2025;15:96567.
Christensen P, Nelson A, Tyler A, Berg K. Development and validation of multiplexed non-human primate ELISA assays. J Immunol. 2023.
Leng SX, McElhaney JE, Walston JD, Xie D, Fedarko NS, Kuchel GA. ELISA and multiplex technologies for cytokine measurement in inflammation and aging research. J Gerontol A Biol Sci Med Sci. 2008;63(8):879–884.
Lewczuk P, Riederer P, O'Bryant SE, Verbeek MM, Dubois B, Visser PJ, et al. Cerebrospinal fluid and blood biomarkers for neurodegenerative dementias: An update of the consensus of the Task Force on Biological Markers in Psychiatry of the World Federation of Societies of Biological Psychiatry. World J Biol Psychiatry. 2018;19(4):244–328
Ungaro CT, Wolfe AS, Brown SD. Comparative analysis of serum cytokine ELISA and multiplex techniques. Biomed J Sci Tech Res. 2020;32(5):25325–25330
Liao T, Yuan F, Yu H, Li Z. An ultrasensitive ELISA method for the detection of procalcitonin based on magnetic beads and enzyme antibody labeled gold nanoparticles. Anal Methods. 2016;8:1577–1585
Feng F, Thompson MP, Thomas BE, Duffy ER, Kim J, Kurosawa S, et al. A computational solution to improve biomarker reproducibility during long term projects. bioRxiv. 2018.
Bozkurt TE. Preklinik çalışmaların kliniğe yansıması ve translasyonel araştırmalar. In: Özet A, Tunçok Y, Özdemir N, editors. Klinik Araştırmalar. 1st ed. Türkiye Klinikleri; 2022. p. 183–186.
Kinders R, Ferry Galow K, Wang L, Srivastava AK, Ji JJ, Parchment RE. Implementation of validated pharmacodynamic assays in multiple laboratories: Challenges, successes, and limitations. Clin Cancer Res. 2014;20(10):2578–2586
Aydin S, Emre E, Ugur K, Aydin MA, Sahin İ, Cinar V, Akbulut T. An overview of ELISA: A review and update on best laboratory practices for quantifying peptides and proteins in biological fluids. J Int Med Res. 2025;53(2):3000605251315913
Marcovina S, Navabi N, Allen S, Gonen A, Witztum J, Tsimikas S. Development and validation of an isoform-independent monoclonal antibody–based ELISA for measurement of lipoprotein(a). J Lipid Res. 2022;63:100239
Percie du Sert N, Hurst V, Ahluwalia A, Alam S, Avey MT, Baker M, et al. The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research. Br J Pharmacol. 2020;177(16):3617–3624.
Gosselin RD. Insufficient transparency of statistical reporting in preclinical research: A scoping review. Sci Rep. 2021;11(1):3335.
World Health Organization. WHO manual for the preparation of secondary reference materials for in vitro diagnostic assays designed for infectious disease nucleic acid or antigen detection: Calibration to WHO International Standards, Annex 6, TRS No. 1004. World Health Organization; 2017
Sluis AR, Padilla A, Das RG. Biological standardization of cytokines and growth factors. Dev Biol Stand. 1999;97:171–176.
MacNeill R, Hutchinson T, Acharya V, Stromeyer R, Ohorodnik S. An oligonucleotide bioanalytical LC SRM methodology entirely liberated from ion pairing. Bioanalysis. 2019;11(12):1157–1169.
Llibre A, Bondet V, Rodero MP, Hunt D, Crow YJ, Duffy D. Development and validation of an ultrasensitive single molecule array digital enzyme-linked immunosorbent assay for human interferon-α. J Vis Exp. 2018;(136):57421
Rissin DM, Kan C, Campbell T, Howes S, Fournier D, Song L. Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations. Nat Biotechnol. 2010;28(6):595–599
Pillai-Kastoori L, Schutz-Geschwender A, Harford J. A systematic approach to quantitative Western blot analysis. Anal Biochem. 2020;113608.
Schägger H, von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987;166(2):368-79
Kurien BT, Scofield RH. Western blotting. Methods. 2006;38(4):283-93
Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979;76(9):4350-4.
Mahmood T, Yang PC. Western blot: technique, theory, and trouble shooting. N Am J Med Sci. 2012;4(9):429-34.
Singh KK, Gupta A, Bharti C, et al. Emerging techniques of western blotting for purification and analysis of protein. Futur J Pharm Sci. 2021;7:239
Horowitz PM, Lee JC, Williams GA, Williams RF, Barnes LD. Electrophoresis of proteins and nucleic acids on acrylamide-agarose gels lacking covalent crosslinking. Anal Biochem. 1984;143(2):333-40.
Garić D, Dumut D, Centorame A, Radzioch D. Western blotting with fast SDS‐PAGE and semi‐dry protein transfer. Curr Protoc. 2023;3.
Zubair M, Launico MV. Western blot: principles, procedures, and clinical applications. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025 Jan-. Updated 2025 Dec 1. Available from: https://www.ncbi.nlm.nih.gov/books/NBK542290
Taylor SC, Berkelman T, Yadav G, et al. A defined methodology for reliable quantification of Western blot data. Mol Biotechnol. 2013;55:217-26.
Yukhananov R, Chimento D, Marlow L. Western blot processing optimization: the perfect blot. Methods Mol Biol. 2021;2349:65–80
Mahmood T, Yang PC. Western blot: technique, theory, and trouble shooting. N Am J Med Sci. 2012;4(9):429–434.
Zeng F, Marino S, Idris A. Analysis of signaling pathways by Western blotting and immunoprecipitation. Methods Mol Biol. 2025;2885:195–208.
Burnette WN. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate–polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981;112(2):195–203. doi:10.1016/0003-2697(81)90281-5
Britz J, Rolon N, Hill T, Page E, Geltosky J. Interpreting HIV ELISA reactivity: alternatives to western blot. J Clin Lab Anal. 1988;2:186–190
Carlton L, McGregor R, Moreland N. Human antibody profiling technologies for autoimmune disease. Immunol Res. 2023;1–12. doi:10.1007/s12026-023-09362-8
Pacoova Dal Maschio V, Roveta F, Bonino L, Boschi S, Rainero I, Rubino E. The role of blood based biomarkers in transforming Alzheimer’s disease research and clinical management: a review. Int J Mol Sci. 2025;26(17):8564.
Noble JE, Bailey MJ. Quantitation of protein. Methods Enzymol. 2009; 463:73–95
Wilson K, Walker J. Principles and Techniques of Biochemistry and Molecular Biology. 7th ed. Cambridge: Cambridge University Press; 2010.
Westermeier R, Naven T, Höpker HR. Proteomics in Practice: A Guide to Successful Experimental Design. 2nd completely revised ed. Weinheim: Wiley-Blackwell; 2008. 502 p
Sapan CV, Lundblad RL. Review of methods for determination of total protein and peptide concentration in biological samples. Proteomics Clin Appl. 2015;9(3–4):268–276.
Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72:248–254
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193(1):265–275.
Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985;150(1):76–85
Crowther JR. ELISA. Theory and practice. Methods Mol Biol. 1995; 42:1–218
Scopes RK. Protein purification: principles and practice. 3rd ed. New York: Springer-Verlag; 1994
Taylor SC, Posch A. The design of a quantitative western blot experiment. Biomed Res Int. 2014; 2014:361590
Vaux DL, Fidler F, Cumming G. Replicates and repeats—what is the difference and is it significant? A brief discussion of statistics and experimental design. EMBO Rep. 2012;13(4):291–296.
Stoscheck CM. Quantitation of protein. Methods Enzymol. 1990; 182:50–68.
Sapan CV, Lundblad RL, Price NC. Colorimetric protein assay techniques. Biotechnol Appl Biochem. 1999;29(2):99–108
Tomimatsu K, Fujii T, Bise R, et al. Precise immunofluorescence canceling for highly multiplexed imaging to capture specific cell states. Nat Commun. 2024;15:3657
Suresh A, Pallaprolu N, Dande A, Pogula H, Parihar V, Peraman R. Temperature- and time-dependent degradation of mouse tissue proteins: insights into RNA-binding protein stability via mass spectrometry. Mol Omics. 2025
Hartl J, Kurth F, Kappert K, et al. Quantitative protein biomarker panels: a path to improved clinical practice through proteomics. EMBO Mol Med. 2023;15:EMMM202216061
Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144(5):646–674.
Zhang X, Wang J, Zhang Z, et al. Tau in neurodegenerative diseases: molecular mechanisms, biomarkers, and therapeutic strategies. Transl Neurodegener. 2024;13:40
Luster A. Chemokines—chemotactic cytokines that mediate inflammation. N Engl J Med. 1998;338(7):436–445
American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care. 2014;37 Suppl 1:S81–S90.
Thygesen K, Alpert JS, Jaffe AS, Chaitman BR, Bax JJ, Morrow DA, White HD, et al. Fourth Universal Definition of Myocardial Infarction (2018). J Am Coll Cardiol. 2018;72(18):2231–2264.
Kraus VB. Biomarkers as drug development tools: discovery, validation, qualification and use. Nat Rev Rheumatol. 2018;14(6):354–362.
Szklarczyk D, Gable AL, Lyon D, Junge A, Wyder S, Huerta-Cepas J, Simonovic M, Doncheva NT, Morris JH, Bork P, Jensen LJ, von Mering C. STRING v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nucleic Acids Res. 2019;47(D1):D607–D61
Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, Amin N, Schwikowski B, Ideker T. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003;13(11):2498–2504.
