Skip to main content
SpringerLink
Log in

Rapid Detection of Femtogram Amounts of Protein by Gel-Free Immunoblot

  • Published:
Bulletin of Experimental Biology and Medicine Aims and scope

The article presents a new method of immunoblotting for simple, rapid, and highly sensitive detection of proteins. Electrophoretic separation of sample is carried out under non-denaturing conditions in a thin conductive layer between cellulose membranes without polyacrylamide gel. The membrane surface is preliminarily modified with azidophenyl groups to photochemically immobilize proteins in situ. For visualization of protein bands, the membranes are treated with magnetic beads coated with specific antibodies, unbound particles are then removed with a magnet. The detection limit in the model system with biotinylated BSA and magnetic beads coated with streptavidin reaches 10 fg or about 105 molecules, while the total blotting time does not exceed 5 min. The method was applied for detection of IgA in a sample of human exhaled air. The method can be used for the analysis of various complex biological samples containing low amounts of the analyte.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Abdel-Sayed P, Yamauchi KA, Gerver RE, Herr AE. Fabrication of an open microfluidic device for immunoblotting. Anal. Chem. 2017;89(18):9643-9648.

    CAS  PubMed  PubMed Central  Google Scholar 

  2. He M, Herr A.E. Automated microfluidic protein immunoblotting. Nat. Prot. 2010;5(11):1844-1856.

    CAS  Google Scholar 

  3. Mikheev AY, Morozov VN. AFM imaging of exhaled microdroplets and dry residues collected by impactor. J. Aerosol Sci. 2018;123:131-140.

    CAS  Google Scholar 

  4. Mikheev AY, Shlyapnikov YM, Kanev IL, Morozov VN, Avseenko AV. Filtering and optical properties of free standing electrospun nanomats from nylon-4,6. Eur. Pol. J. 2016;75:317-328.

    CAS  Google Scholar 

  5. Mishra M, Tiwari S, Gunaseelan A, Li D, Hammock BD, Gomes AV. Improving the sensitivity of traditional Western blotting via Streptavidin containing Poly-horseradish peroxidase (PolyHRP). Electrophoresis. 2019;40(12-13):1731-1739.

  6. Morozov V.N, Morozova T.Y. Active bead-linked immunoassay on protein microarrays. Anal. Chim. Acta. 2006;564(1):40- 52.

    CAS  PubMed  Google Scholar 

  7. Morozov VN, Nikolaev AA, Shlyapnikov YM, Mikheev AY, Shlyapnikova EA, Bagdasaryan TR, Burmistrova IA, Smirnova TG, Andrievskaya IY, Larionova EE, Nikitina IY, Lyadova IV. Non-invasive approach to diagnosis of pulmonary tuberculosis using microdroplets collected from exhaled air. J. Breath Res. 2018;12(3):036010. https://doi.org/10.1088/1752-7163/aab3f2

  8. Morozov VN, Shlyapnikov YM, Kidd J, Morozova TY, Shlyapnikova EA. Conic electrophoretic concentrator for charged macromolecules. Anal. Chem. 2011;83(14):5548-5555.

    CAS  PubMed  Google Scholar 

  9. Shlyapnikov YM, Kanev IL, Shlyapnikova EA. Rapid ultrasensitive gel-free immunoblotting with magnetic labels. Anal. Chem. 2020;92(5):4146-4153.

    CAS  PubMed  Google Scholar 

  10. Shlyapnikov YM, Morozov VN. Titration of trace amounts of immunoglobulins in a microarray-based assay with magnetic labels. Anal. Chim. Acta. 2017;966):47-53.

    CAS  Google Scholar 

  11. Shlyapnikov YM, Shlyapnikova EA, Morozov VN. Carboxymethylcellulose film as a substrate for microarray fabrication. Anal. Chem. 2014;86(4):2082-2089.

    CAS  Google Scholar 

  12. Treindl F, Ruprecht B, Beiter Y, Schultz S, Döttinger A, Staebler A, Joos TO, Kling S, Poetz O, Fehm T, Neubauer H, Kuster B, Templin MF. A bead-based western for highthroughput cellular signal transduction analyses. Nat. Commun. 2016;7:12852. https://doi.org/10.1038/ncomms12852.

Download references

Author information

Authors and Affiliations

  1. Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow region, Russia

    Y. M. Shlyapnikov, E. A. Malakhova & E. A. Shlyapnikova

Authors
  1. Y. M. Shlyapnikov
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. E. A. Malakhova
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. E. A. Shlyapnikova
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Y. M. Shlyapnikov.

Additional information

Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 169, No. 6, pp. 788-792, June, 2020

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Shlyapnikov, Y.M., Malakhova, E.A. & Shlyapnikova, E.A. Rapid Detection of Femtogram Amounts of Protein by Gel-Free Immunoblot. Bull Exp Biol Med 169, 840–843 (2020). https://doi.org/10.1007/s10517-020-04988-2

Download citation

  • Received:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10517-020-04988-2

Key Words

Search

Navigation