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US 2005/0032078 A1Published application
Methods for the detection, analysis and isolation of nascent proteins
AssigneeAmberGen, Inc.
InventorsKenneth Rothschild, Sadanand Gite, Jerzy Olejnik
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Abstract
This invention relates to non-radioactive markers that facilitate the detection and analysis of nascent proteins translated within cellular or cell-free translation systems. Nascent proteins containing these markers can be rapidly and efficiently detected, isolated and analyzed without the handling and disposal problems associated with radioactive reagents. Preferred markers are dipyrrometheneboron difluoride (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) dyes.
Claims37
A reaction mixture, comprising:
- a) a first oligonucleotide primer comprising i) a sequence corresponding to a promoter, ii) a sequence corresponding to a ribosome binding site, iii) a start codon, and iv) a sequence coding for a first epitope marker;
- and
- b) a second oligonucleotide primer comprising i) at least one stop codon, and ii) a sequence encoding for a second epitope marker.
A kit, comprising:
- a) a first oligonucleotide primer comprising i) a sequence corresponding to a promoter, ii) a sequence corresponding to a ribosome binding site, iii) a start codon, and iv) a sequence coding for a first epitope marker;
- and
- b) a second oligonucleotide primer comprising i) at least one stop codon, and ii) a sequence encoding for a second epitope marker.
A method of introducing coding sequence for epitope markers into nucleic acid, comprising:
- a) providing:
- i) a first oligonucleotide primer comprising i) a sequence corresponding to a promoter, ii) a sequence corresponding to a ribosome binding site, iii) a start codon, and iv) a sequence coding for a first epitope marker;
- ii) a second oligonucleotide primer comprising i) at least one stop codon, and ii) a sequence encoding for a second epitope marker;
- iii) a polymerase;
- and
- iv) template;
- and
- b) mixing said template with said first primer, second primer and said polymerase under conditions such that amplified template is produced, said amplified template coding for said first and second epitope markers.
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Dependencies are inferred from the claim text.