Reactive peptide labeling
AssigneePromega Corporation
InventorsHui Wang, Nidhi Nath, Rod Flemming, Becky Godat, Wenhui Zhou, Virginia Kincaid, Melanie Dart
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Abstract
Provided herein are sulfo n-hydroxysuccimidyl ester (sulfo-SE) linked peptides, methods of synthesis thereof, and methods of using such peptides for labeling of biomolecules. In particular, peptides comprising non-alkyl group such as serine, threonine, cysteine, tyrosine, glutamic acid, and aspartic acid are stably modified (e.g., without autoreactivity) with a sulfo-SE group and used to label or otherwise modify biomolecules.
Claims64
A composition comprising a peptide linked to a sulfo n-hydroxysuccimidyl ester (sulfo-SE) group, wherein the peptide does not comprise a cysteine or lysine residue.
The composition of 22, wherein the peptide comprises fluorophore conjugated to an arginine.
The composition of 23, wherein the peptide comprises fluorophore conjugated to SEQ ID NO:
- 23.
The composition of 23, wherein the peptide comprises fluorophore conjugated to SEQ ID NO:
- 10.
A method of labeling a peptide with a sulfo-SE moiety comprising contacting the peptide with a sulfo-NHS compound under conditions such that the hydroxy of the sulfo-NHS compound reacts with the terminal amine of the peptide, wherein the peptide does not comprise a cysteine or lysine residue.
The method of claim 35, wherein the analyte is linked to a complementary polypeptide capable of forming a bioluminescent complex with the peptide on the biomolecule.
A method comprising
- (a) forming a bioluminescent complex of a SEQ ID NO:
- 1 (SmBiT) labeled-analyte biomolecule and the LgBiT-labeled analyte biomolecule specific antibody;
- (b) contacting the bioluminescent complex with the analyte;
- (c) contacting the bioluminescent complex with a substrate for the bioluminescent complex;
- and
- (d) detecting light output from the bioluminescent complex.
A method comprising
- (a) contacting an analyte with a SEQ ID NO:
- 1 (SmBiT) labeled-analyte specific antibody and a LgBiT-labeled analyte-specific antibody, and forming a bioluminescent complex;
- (b) contacting the bioluminescent complex with a substrate for the bioluminescent complex;
- and
- (c) detecting light output from the bioluminescent complex.
A method comprising:
- (a) contacting an analyte with a SEQ ID NO:
- 1 (SmBiT) labeled-analyte specific antibody, a SEQ ID NO:
- 11 (HiBiT)-labeled analyte-specific antibody, and a polypeptide copable for forming a bioluminescent complex with HiBiT and SmBiT;
- (b) contacting the bioluminescent complex with a substrate for the bioluminescent complex;
- and
- (c) detecting light output from the bioluminescent complex.
A method wherein the analyte biomolecule or analyte specific antibody is labeled with fluorophore-conjugated SEQ ID NO:
- 10 (SmBiT) or SEQ ID NO:
- 11 (HiBiT), wherein
- (a) SEQ ID NO:
- 10 (SmBiT) or SEQ ID NO:
- 11 (HiBiT)-labeled-analyte biomolecule contacts the SEQ ID NO:
- 11 (HiBiT) or SEQ ID NO:
- 10 (SmBiT)-labeled analyte biomolecule specific antibody, wherein one of the peptides is the fluorophore-conjugated peptide;
- (b) contacting with LgBiT to form a bioluminescent complex;
- (c) contacting with the analyte
- (d) contacting the bioluminescent complex with a substrate for the bioluminescent complex;
- and
- (e) detecting light output from the bioluminescent complex.
Dependencies are inferred from the claim text.