Life Technologies Corporation

University of Oregon

George Hanson

Adam Michael Zahner

Laurel Rebecca Stone

Zenia Wei

Scott Rickes

Michael O'Grady

Lubasha Chabansky

Paul Boeschoten

Nelson Au

Laurence Trigg

Terence Tam

Erik Persmark

Steven Lytle

Timothy Updyke

Thomas Diller

S James Remington

Bethany Sutton

Joseph Amshey

Kim Ippolito

Roumen Bogoev

Jonathan Young

Nicholas Fargher

Kyle Miller

Microscope system display screen with graphical user interface

Cell counter display screen with graphical user interface

The present invention provides methods and compositions for labeling, separating and analyzing proteins, particularly a specific protein of interest within a cell lysate or in a mixture of proteins. The proteins are labeled with an amine reactive or thiol reactive fluorescent dye, or an amine reactive fluorogenic reagent that becomes fluorescent upon reacting to amine groups located on the protein. Following the labeling step, the proteins within the mixture can be separated and analyzed. In a further embodiment, a tag binding fluorogenic reagent that can bind to a tag on a tagged protein is added to specifically label the protein of interest.

Rapid protein labeling and analysis

Display screen with graphical user interface

Digital microscrope

Digital microscope

The invention is based on the discovery that certain biarsenical molecules react with specified target sequences, thereby providing a facile means for labeling polypeptides containing the target sequence. The invention is useful in creating stable mammalian cell lines expressing a certain tetracysteine tagged polypeptides, thereby overcoming toxicity associated with native tetracysteine. In addition, the invention allows for orthogonal labeling of polypeptides, thereby allowing for the observation of protein-protein interactions and conformational changes in proteins, for example.

Target sequences for synthetic molecules

Methods, compositions, and kits for labeling tetracysteine-tagged proteins with biarsenical fluorophores with increased specificity, including compositions, methods and kits particularly adapted for labeling of tetracysteine-tagged proteins to be resolved within an electrophoresis gel.

Compositions, methods and kits for biarsenical fluorophore labeling

The disclosure provides proteins that can be used to determine the redox status of an environment (such as the environment within a cell or subcellular compartment). These proteins are green fluorescent protein (GFP) variants (also referred to as redox sensitive GFP (rosGFP) mutants), which have been engineered to have two cysteine amino acids near the chromophore and within disulfide bonding distance of each other. Also provided are nucleic acid molecules that encode rosGFPs, vectors containing such encoding molecules, and cells transformed therewith. The disclosure further provides methods of using the rosGFPs (and encoding molecules) to analyze the redox status of an environment, such as a cell, or a subcellular compartment within a cell. In certain embodiments, both redox status and pH are analyzed concurrently.

Nucleic acids encoding oxidation-reduction sensitive green fluorescent protein variants

Oxidation reduction sensitive green fluorescent protein variants

Growing community of inventors

Eugene, OR, United States of America

George Hanson