Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

SDS-polyacrylamide gel electrophoretic

  • 283 Accesses

  • 25 Citations

Summary

A sodium dodecylsulfate-polyacrylamide electrophoretic method, which in contrast to other biochemical procedures, e. g. differential salt precipitation or ion exchange chromatography and molecular sieve chromatography, is applicable to smallest amounts of protein, is shown to be suitable for the determination of the collagen types from small skin samples, such as routine skin biopsies.

After urea extraction, the tissue samples are cleaved with cyanogen bromide. The resulting CNBr peptides derived from the different α-chains are resolved in 12% SDS-polyacrylamide gels. Densitometric profiles of the gel electrophoretic patterns correspond to the collagen type content of the tissue specimens.

Comparing fetal to adult skin, the higher content of type III collagen in the case of fetal skin can be demonstrated.

Zusammenfassung

Es wird eine Dodecylsulfat-Polyacrylamidgel-elektrophoretische Methode beschrieben, mit welcher der Gehalt an Typ I- und Typ III-Kollagen in kleinen Hautproben, z. B. Probebiopsien, bestimmt werden kann. Im Gegensatz zu anderen biochemischen Methoden, z. B. der Salzpräzipitation oder der Ionenaustausch- und Molekularsiebchromatographie, ist diese Methode zur Darstellung kleinster Peptidmengen geeignet.

Harnstoffextrahierte Gewebeproben werden mit Bromcyan abgebaut. Die hierbei freigesetzten, den einzelnen α-Ketten zugeordneten Bromcyan-Peptide werden in 12%igen Gelen aufgetrennt. Aus den densitometrischen Kurvenverläufen der gelelektrophoretischen Peptidverteilungsmuster wird auf den Kollagentypgehalt der untersuchten Gewebeproben geschlossen.

Beim Vergleich von Fötalhaut mit Erwachsenenhaut kann der höhere Typ III-Gehalt der Fötalhaut dargestellt werden.

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

References

  1. Epstein, H. E., Jr.: [α1(III)]3 human skin collagen. Release by pepsin digestion and preponderance in fetal life. J. Biol. Chem.249, 3225–3231 (1974)

  2. Epstein, H. E., Jr., Munderloh, N. H.: Isolation and characterization of CNBr peptides of human [α1(III)]3 collagen and tissue distribution of [α1(I)]2,α2 and [α1(III)]3 collagens. J. Biol. Chem.250, 9304–9312 (1975)

  3. Fietzek, P. P., Kühn, K.: The primary structure of collagen. Int. Rev. Connect. Tiss. Res.7, 1–60 (1976)

  4. Fujii, T., Kühn, K.: Isolation and characterization of pepsin-treated type III collagen from calf skin. Hoppe-Seyler's Z. Physiol. Chem.356, 1793–1801 (1975)

  5. Laemmli, U. K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T 4. Nature (Lond.)227, 680–685 (1970)

  6. Miller, E.J., Epstein, E.H., Jr., Piez, K.A.: Identification of three genetically distinct collagens by cyanogen bromide cleavage of insoluble human skin and cartilage collagen. Biochem. Biophys. Res. Commun.42, 1024–1029 (1971)

  7. Rauterberg, J.: Characterization of a genetic variant of collagen (type III) from calf aorta. IV. Europ. Symp. Connect. Tissue Res., Padua 1974

  8. Rauterberg, J. Allam, S.: In preparation.

  9. Scott, P. G., Telser, A. G., Veis, A.: Semiquantitative determination of cyanogen bromide peptides of collagen in SDS-polyacrylamide gels. Anal. Biochem.70, 251–257 (1976)

  10. Shapiro, A. L., Vinuela, E., Maizel, J. V.: Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem. Biophys. Res. Commun.28, 815–820 (1967)

  11. Trelstad, R. L., Catanese, V. M., Rubin, D. F.: Collagen fractionation: separation of native types I, II and III by differential precipitation. Anal. Biochem.71, 114–118 (1976)

Download references

Author information

Additional information

Author to whom reprint requests should be addressed

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Weber, L., Meigel, W.N. & Rauterberg, J. SDS-polyacrylamide gel electrophoretic. Arch. Derm. Res. 258, 251–257 (1977). https://doi.org/10.1007/BF00561127

Download citation

Keywords

  • Cyanogen
  • Cyanogen Bromide
  • Fetal Skin
  • Urea Extraction
  • Molecular Sieve Chromatography