Methods for the detection, analysis and isolation of Nascent proteins
AssigneeAmberGen, Inc.
InventorsKenneth J. 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.
Claims26
A method of creating a tRNA/marker conjugate, comprising:
- a) providing i) a tRNA molecule charged with an amino acid and ii) a fluorophore marker, wherein said fluorophore marker comprises the structure set forth in FIG. 19;
- and
- b) reacting said tRNA molecule with said marker to create a tRNA/marker conjugate, wherein the amino acid of said tRNA of said tRNA/marker conjugate is covalently bonded to said marker.
A method of introducing conjugates into a translation system, comprising:
- a) providing i) a translation system ii) a first charged tRNA/marker conjugate comprising a first amino acid covalently bound to a marker, wherein said marker comprises the structure set forth in FIG. 19, and ii) a second charged tRNA/marker conjugate comprising a second amino acid covalently bonded to a marker, wherein said marker comprises the structure set forth in FIG. 19, and wherein said first amino acid is different from said second amino acid;
- and
- b) introducing said first and second conjugates into said translation system.
A kit, comprising:
- a) a first containing means containing at least one component of a protein synthesis system;
- b) a second containing means containing a tRNA/marker conjugate, wherein said marker of said tRNA/marker conjugate comprises the structure set forth in FIG. 19.
A tRNA/marker conjugate, wherein said marker comprises the structure set forth in FIG. 19.
A method of creating a charged tRNA molecule, comprising:
- a) providing i) a tRNA molecule and ii) a conjugate comprising an amino acid covalently bonded to a fluorophore marker, wherein said fluorophore marker comprises the structure set forth in FIG. 19;
- and
- b) creating a charged tRNA molecule with said conjugate.
A method of introducing a conjugate into a translation system, comprising:
- a) providing i) a translation system ii) a charged tRNA/marker conjugate comprising a amino acid covalently bound to a marker, wherein said marker comprises the structure set forth in FIG. 19;
- b) introducing said conjugate into said translation system under conditions such that said marker is incorporated into a nascent protein;
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- c) visualizing said nascent protein on a gel virtue of said marker.
Dependencies are inferred from the claim text.