Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1991 Mar 15;88(6):2563–2567. doi: 10.1073/pnas.88.6.2563

Chemiluminescence detection of proteins from single cells.

P G Gillespie 1, A J Hudspeth 1
PMCID: PMC51273  PMID: 2006191

Abstract

The analysis of proteins from single cells requires techniques of supreme sensitivity. Although radiochemical procedures are capable of detecting small amounts of electrophoretically separated proteins, their sensitivity falls short of that required for routine detection of minor components of single cells. Utilizing the avidin-biotin interaction and the alkaline phosphatase substrate 3-(4-methoxyspiro[1,2-dioxetane-3,2'- tricyclo-[3.3.1.1(3,7)]decan]-4-yl)phenyl phosphate (AMPPD), we have developed an alternative, chemiluminescence-based method for protein detection whose sensitivity exceeds that of other methods. Applying this method to a purified protein, we could detect as little as 63 fg (0.9 amol) of biotinylated bovine serum albumin. The sensitivity of the method was demonstrated by the detection of proteins from individual photoreceptor outer segments, including proteins constituting approximately 1% of the total. Chemiluminescence detection also proved extremely sensitive for immunoblotting: a comparison of five methods for detection of antibody-antigen interactions showed that the AMPPD technique was more sensitive than detection with a colorimetric alkaline phosphatase substrate, 125I-labeled protein A, 125I-labeled anti-mouse IgG, or colloidal gold-conjugated anti-mouse IgG.

Full text

PDF
2563

Images in this article

2565Image
on p.2565
2565Image
on p.2565
2566Image
on p.2566

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bayer E. A., Wilchek M. Protein biotinylation. Methods Enzymol. 1990;184:138–160. doi: 10.1016/0076-6879(90)84268-l. [DOI] [PubMed] [Google Scholar]
  2. Blake M. S., Johnston K. H., Russell-Jones G. J., Gotschlich E. C. A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots. Anal Biochem. 1984 Jan;136(1):175–179. doi: 10.1016/0003-2697(84)90320-8. [DOI] [PubMed] [Google Scholar]
  3. Bolton A. E., Hunter W. M. The labelling of proteins to high specific radioactivities by conjugation to a 125I-containing acylating agent. Biochem J. 1973 Jul;133(3):529–539. doi: 10.1042/bj1330529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brady S. T. A novel brain ATPase with properties expected for the fast axonal transport motor. Nature. 1985 Sep 5;317(6032):73–75. doi: 10.1038/317073a0. [DOI] [PubMed] [Google Scholar]
  5. Bronstein I., Edwards B., Voyta J. C. 1,2-dioxetanes: novel chemiluminescent enzyme substrates. Applications to immunoassays. J Biolumin Chemilumin. 1989 Jul;4(1):99–111. doi: 10.1002/bio.1170040116. [DOI] [PubMed] [Google Scholar]
  6. Bronstein I., McGrath P. Chemiluminescence lights up. Nature. 1989 Apr 13;338(6216):599–600. doi: 10.1038/338599a0. [DOI] [PubMed] [Google Scholar]
  7. Bronstein I., Voyta J. C., Lazzari K. G., Murphy O., Edwards B., Kricka L. J. Rapid and sensitive detection of DNA in Southern blots with chemiluminescence. Biotechniques. 1990 Mar;8(3):310–314. [PubMed] [Google Scholar]
  8. Burnette W. N. "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 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  9. Durrant I. Light-based detection of biomolecules. Nature. 1990 Jul 19;346(6281):297–298. doi: 10.1038/346297a0. [DOI] [PubMed] [Google Scholar]
  10. GREEN N. M. A SPECTROPHOTOMETRIC ASSAY FOR AVIDIN AND BIOTIN BASED ON BINDING OF DYES BY AVIDIN. Biochem J. 1965 Mar;94:23C–24C. doi: 10.1042/bj0940023c. [DOI] [PubMed] [Google Scholar]
  11. Gillespie P. G., Hudspeth A. J. High-purity isolation of bullfrog hair bundles and subcellular and topological localization of constituent proteins. J Cell Biol. 1991 Feb;112(4):625–640. doi: 10.1083/jcb.112.4.625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gray-Keller M. P., Biernbaum M. S., Bownds M. D. Transducin activation in electropermeabilized frog rod outer segments is highly amplified, and a portion equivalent to phosphodiesterase remains membrane-bound. J Biol Chem. 1990 Sep 5;265(25):15323–15332. [PubMed] [Google Scholar]
  13. Hamm H. E., Bownds M. D. Protein complement of rod outer segments of frog retina. Biochemistry. 1986 Aug 12;25(16):4512–4523. doi: 10.1021/bi00364a010. [DOI] [PubMed] [Google Scholar]
  14. Hsu Y. H. Immunogold for detection of antigen on nitrocellulose paper. Anal Biochem. 1984 Oct;142(1):221–225. doi: 10.1016/0003-2697(84)90542-6. [DOI] [PubMed] [Google Scholar]
  15. Hurley W. L., Finkelstein E., Holst B. D. Identification of surface proteins on bovine leukocytes by a biotin-avidin protein blotting technique. J Immunol Methods. 1985 Dec 17;85(1):195–202. doi: 10.1016/0022-1759(85)90287-x. [DOI] [PubMed] [Google Scholar]
  16. Kühn H. Light- and GTP-regulated interaction of GTPase and other proteins with bovine photoreceptor membranes. Nature. 1980 Feb 7;283(5747):587–589. doi: 10.1038/283587a0. [DOI] [PubMed] [Google Scholar]
  17. LaRochelle W. J., Froehner S. C. Immunochemical detection of proteins biotinylated on nitrocellulose replicas. J Immunol Methods. 1986 Aug 21;92(1):65–71. doi: 10.1016/0022-1759(86)90504-1. [DOI] [PubMed] [Google Scholar]
  18. Lisanti M. P., Sargiacomo M., Graeve L., Saltiel A. R., Rodriguez-Boulan E. Polarized apical distribution of glycosyl-phosphatidylinositol-anchored proteins in a renal epithelial cell line. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9557–9561. doi: 10.1073/pnas.85.24.9557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Marchalonis J. J. An enzymic method for the trace iodination of immunoglobulins and other proteins. Biochem J. 1969 Jun;113(2):299–305. doi: 10.1042/bj1130299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Moeremans M., Daneels G., Van Dijck A., Langanger G., De Mey J. Sensitive visualization of antigen-antibody reactions in dot and blot immune overlay assays with immunogold and immunogold/silver staining. J Immunol Methods. 1984 Nov 30;74(2):353–360. doi: 10.1016/0022-1759(84)90303-x. [DOI] [PubMed] [Google Scholar]
  21. Ochs D. Protein contaminants of sodium dodecyl sulfate-polyacrylamide gels. Anal Biochem. 1983 Dec;135(2):470–474. doi: 10.1016/0003-2697(83)90714-5. [DOI] [PubMed] [Google Scholar]
  22. Papasozomenos S. C., Binder L. I. Phosphorylation determines two distinct species of Tau in the central nervous system. Cell Motil Cytoskeleton. 1987;8(3):210–226. doi: 10.1002/cm.970080303. [DOI] [PubMed] [Google Scholar]
  23. Peluso R. W., Rosenberg G. H. Quantitative electrotransfer of proteins from sodium dodecyl sulfate-polyacrylamide gels onto positively charged nylon membranes. Anal Biochem. 1987 May 1;162(2):389–398. doi: 10.1016/0003-2697(87)90409-x. [DOI] [PubMed] [Google Scholar]
  24. Pfister K. K., Wagner M. C., Stenoien D. L., Brady S. T., Bloom G. S. Monoclonal antibodies to kinesin heavy and light chains stain vesicle-like structures, but not microtubules, in cultured cells. J Cell Biol. 1989 Apr;108(4):1453–1463. doi: 10.1083/jcb.108.4.1453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ramírez P., Bonilla J. A., Moreno E., León P. Electrophoretic transfer of viral proteins to nitrocellulose sheets and detection with peroxidase-bound lectins and protein A. J Immunol Methods. 1983 Aug 12;62(1):15–22. doi: 10.1016/0022-1759(83)90105-9. [DOI] [PubMed] [Google Scholar]
  26. Roy S. K., Kundu S. K. Chemically modified porous silica gel as a bioadsorbent and a biocatalyst. Anal Biochem. 1979 Sep 15;98(1):238–241. doi: 10.1016/0003-2697(79)90733-4. [DOI] [PubMed] [Google Scholar]
  27. Schaap A. P., Akhavan H., Romano L. J. Chemiluminescent substrates for alkaline phosphatase: application to ultrasensitive enzyme-linked immunoassays and DNA probes. Clin Chem. 1989 Sep;35(9):1863–1864. [PubMed] [Google Scholar]
  28. Schleicher M., Watterson D. M. Analysis of differences between coomassie blue stain and silver stain procedures in polyacrylamide gels: conditions for the detection of calmodulin and troponin C. Anal Biochem. 1983 Jun;131(2):312–317. doi: 10.1016/0003-2697(83)90175-6. [DOI] [PubMed] [Google Scholar]
  29. Tizard R., Cate R. L., Ramachandran K. L., Wysk M., Voyta J. C., Murphy O. J., Bronstein I. Imaging of DNA sequences with chemiluminescence. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4514–4518. doi: 10.1073/pnas.87.12.4514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Vale R. D., Reese T. S., Sheetz M. P. Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility. Cell. 1985 Aug;42(1):39–50. doi: 10.1016/s0092-8674(85)80099-4. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES