An oligodeoxynucleotide affinity column for the isolation of sequence specific DNA binding proteins.

Abstract:

:A nucleic acid affinity matrix containing a short oligodeoxynucleotide ligand has been prepared as an example of a material which can be used for the rapid and effective isolation of sequence specific DNA binding proteins. Two complementary oligodeoxynucleotides have been employed, one of which contains a small 5'-spacer arm with a terminal thiol group. Using this terminal thiol group, the ligand can be covalently coupled to Tresyl-activated Sepharose 4B or Epoxy-activated Sepharose 6B via a thioether linkage. This approach allows the specific attachment of the nucleic acid ligand via its 5'-terminus to the insoluble matrix. The double stranded affinity material was obtained by annealing of the complementary DNA fragment. As an example, we have used an eicosomer affinity column containing the sequence d(GAATTC) for the isolation of the Eco RI restriction endonuclease. Using a single column, the enzyme could be isolated by eluting the column with a single step or multistep gradient of increasing salt concentration. The enzyme was purified to 75%-85% homogeneity with yields of 0.1 mg to 0.2 mg from 0.5 g of cell paste.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Blanks R,McLaughlin LW

doi

10.1093/nar/16.21.10283

subject

Has Abstract

pub_date

1988-11-11 00:00:00

pages

10283-99

issue

21

eissn

0305-1048

issn

1362-4962

journal_volume

16

pub_type

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