AccScience Publishing / JBM / Online First / DOI: 10.14440/jbm.0429
Submit to JBM
Cite this article
1
Download
11
Views
Journal Browser
Volume | Year
Issue
Search
Forthcoming Issue
Current Issue
View All
News and Announcements
View All
PROTOCOL

An improved slot blot assay for protein quantification in serum samples

Abby Kegg1† ,  Parth R. Patel2† ,  Brendan A. Hilliard1 ,  Megan Van Der Bas2 ,  Betsy A. Kallicharan1 ,  Khyleisha A. Ceasar1 ,  Astha Rajpal1 ,  Mikhail A. Kolpakov1 ,  Mary F. Barbe1*
Show Less
1 Department of Cardiovascular Sciences, Aging and Cardiovascular Discovery Center, Lewis Katz School of Medicine, Temple University, Philadelphia 19140, Pennsylvania, United States of America
2 Biomedical Science Graduate Program, Lewis Katz School of Medicine, Temple University, Philadelphia 19140, Pennsylvania, United States of America
JBM, 99010102 https://doi.org/10.14440/jbm.0429
Submitted: 10 December 2025 | Revised: 6 February 2026 | Accepted: 10 February 2026 | Published: 29 April 2026
© 2026 by the Author(s). This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution -Noncommercial 4.0 International License (CC-by the license) ( https://creativecommons.org/licenses/by-nc/4.0/ )
Download PDF
Cite
XML
Abstract

Background: Enzyme-linked immunosorbent assay (ELISA) is the current preferred method for analyzing serum samples; however, it is resource-intensive, and serum samples have classically posed challenges for traditional immunoblot-based assays due to interference from serum proteins. This may be attributed to the non-specific binding between secondary antibodies and immunoglobulin G (IgGs) in the serum. Objective: To establish an improved slot blot assay protocol that eliminates non-specific staining for an effective analysis of proteins in serum samples. Methods: We explored the utility of Protein G–agarose beads for depleting serum IgGs prior to slot blot analysis, comparing 1–2 rounds of IgG clearance with no clearance. Serum samples were subsequently analyzed for the relative abundance of four target proteins using cleared serum and slot blotting. Densitometric analysis of immunodetected protein bands was performed and normalized to total protein loading, as determined by Ponceau S red staining of membranes. Slot blot results were compared to ELISA-detected levels. Results: Optimal results were achieved with two rounds of serum incubation with Protein G–agarose beads to deplete IgGs prior to slot blotting. This method eliminated non-specific signals in membranes probed with secondary antibody alone and enabled the specific detection of serum proteins with primary antibodies. Spearman’s rank correlations showed moderate to strong correlations between slot blots and ELISAs for each protein examined. Conclusion: The addition of Protein G–agarose-mediated IgG depletion permits the reliable use of slot blots for the analysis of serum proteins.

Keywords
Protein chemistry
Immunoblotting
Slot blot
Dot blot
Immunodetection assay validation
Funding
This study was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) of the National Institutes of Health (NIH) under Award Number AR056019 to MFB. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The study sponsors did not have any involvement in the study design, data collection, analysis, or interpretation, the writing of the report, or the decision to submit the paper for publication.
Conflict of interest
The authors declare they have no competing interests.
References
  1. Brown T. Dot and slot blotting of DNA. Curr Protoc Mol Biol. 2001;Chapter 2:Unit 2.9B. doi: 10.1002/0471142727.mb0209bs21

 

  1. Bio-Rad. Bio-Dot® SF Microfiltration Apparatus Instruction Manual. Hercules, CA: Bio-Rad. Available from: https://www. bio-rad.com/webroot/web/pdf/lsr/literature/M1706542.pdf [Last accessed on 2026 Feb 26].

 

  1. Brown T. Analysis of RNA by northern and slot-blot hybridization. Curr Protoc Immunol. 2001;Chapter 10:Unit 10.12. doi: 10.1002/0471142735.im1012s07

 

  1. Sarker AH, Cooper PK. Slot Blot Assay for Detection of R Loops. In: Bhakat KK, Hazra TK, eds. Base Excision Repair Pathway: Methods and Protocols. Springer US; 2023:149- 156. doi: 10.1007/978-1-0716-3373-9_4

 

  1. Ramirez P, Crouch RJ, Cheung VG, Grunseich C. R-Loop Analysis by Dot-Blot. J Vis Exp. 2021;(167):e62069. doi: 10.3791/62069

 

  1. beldenwj. Slot/Dot Blot protocol. protocols.io. Published September 8, 2020. Available from: https://www.protocols.io/ view/slot-dot-blot-protocol-8epv5z7d4v1b/v1 [Last accessed on 2026 Feb 05].

 

  1. StressMarq Biosciences Inc. Protocols|Dot Blot. StressMarq Biosciences Inc. Available from: https://www.stressmarq.com/ support/technical-support/protocols/dot-blot-protocol [Last accessed on 2026 Feb 04].

 

  1. Takata T. Is the Novel Slot Blot a Useful Method for Quantification of Intracellular Advanced Glycation End- Products? Metabolites. 2023;13(4):568. doi: 10.3390/metabo13040568

 

  1. Bronowicka-Szydelko A, Krzystek-Korpacka M, Kuzan A, et al. Non-standard AGE4 epitopes that predict polyneuropathy independently of obesity can be detected by slot dot-blot immunoassay. Adv Clin Exp Med. 2020;29(1):91-100. doi: 10.17219/acem/112612

 

  1. Ameri Z, Vahidi R, Khaleghi M, Dehesh T, Sheikhbardsiri A, Farsinejad A. Slot blotting and flow cytometry: two efficient assays for platelet antibody screening among patients with platelet refractoriness. Vox Sang. 2021;116(1):106-115. doi: 10.1111/vox.12988

 

  1. Zulkarnain Z, Ulhaq ZS, Sujuti H, et al. Comparative performance of ELISA and dot blot assay for TSH-receptor antibody detection in Graves’ disease. J Clin Lab Anal. 2022;36(4):e24288.doi: 10.1002/jcla.24288

 

  1. Tate J, Ward G. Interferences in immunoassay. Clin Biochem Rev. 2004;25(2):105-120.

 

  1. Yadav G. Western Blot Detection without Interfering IgGs: A Mere Fantasy or a Real Possibility? Bio-Radiations. Bio-Rad. Available from: https://www.bioradiations.com/western-blot-detection-without-interfering-iggs-a-mere-fantasy-or-a-real-possibility/#:~:text=How%20many%20times%20have%20 we,IgG%20present%20in%20the%20sample [Last accessed on 2026 Feb 04].

 

  1. Bio-Rad. How to detect your target protein or protein complex without interfering IgG chains. Bio-Rad. Available from: https://www.bio-rad-antibodies.com/blog/how-to-detect-your-target-protein-or-protein-complex-without-interfering-igg-chains.html#:~:text=Immunoprecipitation%20(IP)%20is%20 a%20key,non%2Dspecific%20secondary%20antibody%20 binding [Last accessed on 2026 Feb 05].

 

  1. Patel P, Jamal Z, Ramphul K. Immunoglobulin. In: StatPearls. Treasure Island, FL: StatPearls Publishing; 2025.

 

  1. Biersteker R, Abendstein L, van de Bovenkamp S, et al. Modulation of immunoglobulin G oligomerization by variable domain glycans: A mechanism to regulate complement activation. PNAS Nexus. 2025;4(7):pgaf216. doi: 10.1093/pnasnexus/pgaf216

 

  1. Bio-Rad. Protein Blotting Guide. Bulletin 2695 Ver C. Hercules, CA: Bio-Rad. Available from: https://www.bio-rad. com/webroot/web/pdf/lsr/literature/Bulletin_2895.pdf [Last accessed on 2026 Feb 26].

 

  1. Spadella MA, Moreira RJ, Braga PC, Chies AB, Oliveira PF, Alves MG. Losartan Protects Against Radiation- Induced Testicular Damage by Modulating Oxidative Stress, Testosterone Levels, and Metabolic Profile. Pharmaceuticals. 2025;19(1):76. doi: 10.3390/ph19010076

 

  1. Lee WJ, Keefer K, Hollenbeak CS, Stack BC Jr. A new assay to screen for head and neck squamous cell carcinoma using the tumor marker metallopanstimulin. Otolaryngol Head Neck Surg. 2004;131(4):466-471. doi: 10.1016/j.otohns.2004.03.011

 

  1. Xin DL, Hadrevi J, Elliott ME, et al. Effectiveness of conservative interventions for sickness and pain behaviors induced by a high repetition high force upper extremity task. BMC Neurosci. 2017;18(1):36. doi: 10.1186/s12868-017-0354-3

 

  1. Xin DL, Harris MY, Wade CK, Amin M, Barr AE, Barbe MF. Aging enhances serum cytokine response but not task-induced grip strength declines in a rat model of work-related musculoskeletal disorders. BMC Musculoskelet Disord. 2011;12:63. doi: 10.1186/1471-2474-12-63

 

  1. Barbe MF, Lambi AG, Harris MY, et al. Overuse injury induces persistent behavioral declines that correlate with higher IL-6 expression in the affected musculoskeletal tissues, circulation, and brain. Front Physiol. 2025;16:1500795. doi: 10.3389/fphys.2025.1500795

 

  1. Sensi S, Goebel A. Human Auto-IgG Purification from High Volume Serum Sample by Protein G Affinity Purification. Bio Protoc. 2022;12(23):e4562. doi: 10.21769/BioProtoc.4562

 

  1. Sheng S, Kong F. Separation of antigens and antibodies by immunoaffinity chromatography. Pharm Biol. 2012;50(8):1038-1044. doi: 10.3109/13880209.2011.653493

 

  1. Zarrineh M, Mashhadi IS, Farhadpour M, Ghassempour A. Mechanism of antibodies purification by protein A. Anal Biochem. 2020;609:113909. doi: 10.1016/j.ab.2020.113909

 

  1. Fishman JB, Berg EA. Protein A and Protein G Purification of Antibodies. Cold Spring Harb Protoc. 2019;2019(1):pdb. prot099143. doi: 10.1101/pdb.prot099143

 

  1. Klyne DM, Hilliard BA, Harris MY, et al. Poor sleep versus exercise: A duel to decide whether pain resolves or persists after injury. Brain Behav Immun Health. 2024;35:100714. doi: 10.1016/j.bbih.2023.100714

 

  1. Lewis M. Western Blotting Troubleshooting Guide! Jackson ImmunoResearch Laboratories Inc. Available from: https:// www.jacksonimmuno.com/secondary-antibody-resource/ technical-tips/western-blot-trouble-shooting [Last accessed on 2026 Feb 04].

 

  1. Saper CB. A guide to the perplexed on the specificity of antibodies. J Histochem Cytochem. 2009;57(1):1-5. doi: 10.1369/jhc.2008.952770

 

  1. Lewis M. Cross-Adsorbed Secondary Antibodies and Cross- Reactivity. Jackson ImmunoResearch Laboratories Inc. Available from: https://www.jacksonimmuno.com/secondary-antibody-resource/technical-tips/cross-adsorbed-and-cross-reactivity [Last accessed on 2026 Feb 05].

 

  1. Freeman LA. Western Blots. In: Freeman L, ed. Lipoproteins and Cardiovascular Disease Methods in Molecular Biology. Humana Press; 2013:123-136. doi: 10.1007/978-1-60327-369-5_18

 

  1. Papageorgiou SN. On correlation coefficients and their interpretation. J Orthod. 2022;49(3):359-361. doi: 10.1177/14653125221076142

 

  1. Swinscow TDV. Correlation and regression. BMJ. 1997. Available from: https://www.bmj.com/about-bmj/resources-readers/publications/statistics-square-one/11-correlation-and-regression [Last accessed on 2026 Feb 04].

 

  1. Evans JD. Straightforward Statistics for the Behavioral Sciences. Brooks/Cole Pub. Co.; 1996.

 

  1. Voskuil JLA, Bandrowski A, Begley CG, et al. The Antibody Society’s antibody validation webinar series. MAbs. 2020;12(1):1794421. doi: 10.1080/19420862.2020.1794421

 

  1. Chakafana G, Makumire S, Zininga T, Shonhai A. Exploring the application of ELISA and slot blot as utility protein–protein interaction analysis tools. Trans R Soc S Afr. 2022;77(3):175- 180. doi: 10.1080/0035919X.2022.2144536

 

  1. Kenfe FR, Urbaczek AC, Silva JC, Neo TA, da Silva FH, da Costa PI. Development of diagnostic methods and study of the immunoreactivity of a mixture of recombinant core and E2 proteins fused to GST with control serum positive for hepatitis C. Talanta. 2013;110:32-38. doi: 10.1016/j.talanta.2013.02.017

 

  1. Japolla G, Cunha-Junior JP, Pajuaba A, et al. Development of a nanogold slot blot inhibition assay for the detection of antibodies against bovine herpesvirus type 1. Arch Virol. 2018;163(6):1549-1557. doi: 10.1007/s00705-018-3763-4

 

  1. Iannarone VJ, Cruz GE, Hilliard BA, Barbe MF. The answer depends on the question: Optimal conditions for western blot characterization of muscle collagen type 1 depends on desired isoform. J Biol Methods. 2019;6(3):e117. doi: 10.14440/jbm.2019.289

 

  1. Kang W, Zhang J, Li H, et al. Identification of Major B-Cell Linear Epitopes of Peach Allergen Pru p 3 Using Immune Slot- Blot Microarray Assay. J Agric Food Chem. 2022;70(26):8134- 8144. doi: 10.1021/acs.jafc.2c01448
Previous article in this issue
Next article in this issue
Share
Back to top
Journal of Biological Methods, Electronic ISSN: 2326-9901 Print ISSN: TBA, Published by POL Scientific
We use cookies on our website to ensure you get the best experience.
Read more about our cookies here
Accept