Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1980 Jan 25;8(2):299–317. doi: 10.1093/nar/8.2.299

Electrophoretic transfer of DNA, RNA and protein onto diazobenzyloxymethyl (DBM) - paper.

E J Stellwag, A E Dahlberg
PMCID: PMC327268  PMID: 6158735

Abstract

A method has been developed for the electrophoretic transfer of DNA, RNA, protein and ribonucleoprotein particles from a variety of gels onto diazobenzyloxymethyl (DBM) - paper. Conditions for the electrophoretic transfer of these macromolecules have been optimized to allow for nearly quantitative transfer and covalent coupling. DNA and RNA electrophoretically transferred to DBM-paper retain their ability to hybridize with specific probes. The high efficiency of transfer and the high capacity of DBM-paper for nucleic acids makes possible the sensitive detection of specific nucleotide sequences. Similar efficiency is achieved in electrophoretic transfer and covalent coupling of proteins to DBM-paper. Macromolecules can also be electrophoretically transferred and bound to DBM-paper incapable of covalent bond formation. Their elution from the paper in high salt provides a new and useful preparative method for isolation of DNA, RNA and protein.

Full text

PDF
299

Images in this article

305Image
on p.305
309Image
on p.309
310Image
on p.310
310Image
on p.310
311Image
on p.311
312Image
on p.312
313Image
on p.313

Selected References

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

  1. Alwine J. C., Kemp D. J., Stark G. R. Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5350–5354. doi: 10.1073/pnas.74.12.5350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Clewell D. B., Helinski D. R. Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an opern circular DNA form. Proc Natl Acad Sci U S A. 1969 Apr;62(4):1159–1166. doi: 10.1073/pnas.62.4.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Clewell D. B. Nature of Col E 1 plasmid replication in Escherichia coli in the presence of the chloramphenicol. J Bacteriol. 1972 May;110(2):667–676. doi: 10.1128/jb.110.2.667-676.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dahlberg A. E., Dingman C. W., Peacock A. C. Electrophoretic characterization of bacterial polyribosomes in agarose-acrylamide composite gels. J Mol Biol. 1969 Apr 14;41(1):139–147. doi: 10.1016/0022-2836(69)90131-4. [DOI] [PubMed] [Google Scholar]
  5. Dahlberg A. E., Peacock A. C. Studies of 16 and 23 S ribosomal RNA of Escherichia coli using composite gel electrophoresis. J Mol Biol. 1971 Jan 14;55(1):61–74. doi: 10.1016/0022-2836(71)90281-6. [DOI] [PubMed] [Google Scholar]
  6. Free S. J., Rice P. W., Metzenberg R. L. Arrangement of the genes coding for ribosomal ribonucleic acids in Neurospora crassa. J Bacteriol. 1979 Mar;137(3):1219–1226. doi: 10.1128/jb.137.3.1219-1226.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Howard G. A., Traut R. R. A modified two-dimensional gel system for the separation and radioautography of microgram amounts of ribosomal proteins. Methods Enzymol. 1974;30:526–539. doi: 10.1016/0076-6879(74)30052-3. [DOI] [PubMed] [Google Scholar]
  8. Ikemura T., Dahlberg J. E. Small ribonucleic acids of Escherichia coli. I. Characterization by polyacrylamide gel electrophoresis and fingerprint analysis. J Biol Chem. 1973 Jul 25;248(14):5024–5032. [PubMed] [Google Scholar]
  9. Kim K. T., Apirion D., Ghora B. K. Small stable RNA of Neurospora crassa. Mol Gen Genet. 1978 Mar 20;160(1):25–32. doi: 10.1007/BF00275115. [DOI] [PubMed] [Google Scholar]
  10. Kindler P., Keil T. U., Hofschneider P. H. Isolation and characterization of a ribonuclease 3 deficient mutant of Escherichia coli. Mol Gen Genet. 1973 Oct 16;126(1):53–59. doi: 10.1007/BF00333481. [DOI] [PubMed] [Google Scholar]
  11. LEBOY P. S., COX E. C., FLAKS J. G. THE CHROMOSOMAL SITE SPECIFYING A RIBOSOMAL PROTEIN IN ESCHERICHIA COLI. Proc Natl Acad Sci U S A. 1964 Dec;52:1367–1374. doi: 10.1073/pnas.52.6.1367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  13. Landy A., Abelson J., Goodman H. M., Smith J. D. Specific hybridization of tyrosine transfer ribonucleic acids with DNA from a transducing bacteriophage phi-80 carrying the amber suppressor gene su 3. J Mol Biol. 1967 Nov 14;29(3):457–471. doi: 10.1016/0022-2836(67)90112-x. [DOI] [PubMed] [Google Scholar]
  14. Lindahl L., Forchhammer J. Evidence for reduced breakdown of messenger RNA during blocked transcription or translation in Escherichia coli. J Mol Biol. 1969 Aug 14;43(3):593–606. doi: 10.1016/0022-2836(69)90361-1. [DOI] [PubMed] [Google Scholar]
  15. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Noyes B. E., Stark G. R. Nucleic acid hybridization using DNA covalently coupled to cellulose. Cell. 1975 Jul;5(3):301–310. doi: 10.1016/0092-8674(75)90105-1. [DOI] [PubMed] [Google Scholar]
  17. Peacock A. C., Dingman C. W. Molecular weight estimation and separation of ribonucleic acid by electrophoresis in agarose-acrylamide composite gels. Biochemistry. 1968 Feb;7(2):668–674. doi: 10.1021/bi00842a023. [DOI] [PubMed] [Google Scholar]
  18. Rave N., Crkvenjakov R., Boedtker H. Identification of procollagen mRNAs transferred to diazobenzyloxymethyl paper from formaldehyde agarose gels. Nucleic Acids Res. 1979 Aug 10;6(11):3559–3567. doi: 10.1093/nar/6.11.3559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Reiser J., Renart J., Stark G. R. Transfer of small DNA fragments from polyacrylamide gels to diazobenzyloxymethyl-paper and detection by hybridization with DNA probes. Biochem Biophys Res Commun. 1978 Dec 14;85(3):1104–1112. doi: 10.1016/0006-291x(78)90656-3. [DOI] [PubMed] [Google Scholar]
  20. Renart J., Reiser J., Stark G. R. Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3116–3120. doi: 10.1073/pnas.76.7.3116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  22. Roychoudhury R., Jay E., Wu R. Terminal labeling and addition of homopolymer tracts to duplex DNA fragments by terminal deoxynucleotidyl transferase. Nucleic Acids Res. 1976 Apr;3(4):863–877. doi: 10.1093/nar/3.4.863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sharp P. A., Sugden B., Sambrook J. Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose--ethidium bromide electrophoresis. Biochemistry. 1973 Jul 31;12(16):3055–3063. doi: 10.1021/bi00740a018. [DOI] [PubMed] [Google Scholar]
  24. Stark G. R., Williams J. G. Quantitative analysis of specific labelled RNA'S using DNA covalently linked to diazobenzyloxymethyl-paper. Nucleic Acids Res. 1979 Jan;6(1):195–203. doi: 10.1093/nar/6.1.195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Wahl G. M., Stern M., Stark G. R. Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3683–3687. doi: 10.1073/pnas.76.8.3683. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES