Protein Labeling & Conjugation
Labeled proteins allow us to study specific molecular interactions with high sensitivity in complex biological systems. They are important reagents in numerous biological applications such as assays, purifications, protein arrays, localization studies, flow cytometry, clinical imaging and much more. The quality of labeled proteins is critical for consistent and reliable data. Although labeling procedures appear to be simple and straightforward, most of them still need to be adjusted to take into consideration the nature of a protein in order to achieve the desired results. Possible problems during labeling procedures include protein losses due to precipitation, sample manipulation & instability, inconsistent label-to-protein ratio, incomplete removal of an unconjugated labeling probe, and poor protein characterization before and after labeling. In addition, new labeling technologies had emerged for site-directed labeling requiring an integration of protein expression and purification into the labeling process. We have extensive experience with various protein labeling techniques and are confident that we can provide you with high-quality labeled reagents for your downstream applications.
NONSELECTIVE PROTEIN LABELING
- biotinylation with incorporation ratios determined by a HABA-based assay
- fluorescent probe conjugation with incorporation ratios determined by fluorescence
- other chemical moieties (for example, Sulfo-Tag, Dyes) with incorporation ratios determined by UV-Vis spectroscopy
- enzyme conjugation with incorporation ratios determined by enzyme activity assays
- C-terminal labeling through "Sortagging"
- N-terminal labeling through "Sortagging"
- labeling at glycosylation sites
CLICK CHEMISTRY LABELING
- azido modified proteins and/or glycochains
- alkyne modified proteins and/or glycochains
- copper (I)-catalyzed click labeling
- copper-free click labeling
- various commercial choices of click partners including dual labels
- in situ applications
- protein conjugation and detection
BIOMOLECULE IMMOBILIZATION ON SOLID SUPPORT
- site-specific immobilization on a chip in oriented fashion
- protein conjugation to chromatography resin
Useful Tools for Protein Labeling & Conjugation ProjectProtein Labeling Calculator
Degree of Labeling Calculator
Selected Latest Developments in Protein Labeling & Conjugation
The Ongoing Quest to Crack the Genetic Code for Protein Production.Thijs Nieuwkoo et al., Mol Cell, 2020 Oct 15;80(2):193-209.
Protein labeling methods prior to separation and analysis have become indispensable approaches for proteomic profiling. Basically, three different types of tags are employed: stable isotopes, mass tags, and fluorophores. While proteins labeled with stable isotopes and mass tags are measured and differentiated by mass spectrometry, fluorescent labels are detected with fluorescence imagers. The major purposes for protein labeling are monitoring of biological processes, reliable quantification of compounds and specific detection of protein modifications and isoforms in multiplexed samples, enhancement of detection sensitivity, and simplification of detection workflows. Proteins can be labeled during cell growth by incorporation of amino acids containing different isotopes, or in biological fluids, cells or tissue samples by attaching specific groups to the ε-amino group of lysine, the N-terminus, or the cysteine residues. The principles and the modifications of the different labeling approaches on the protein level are described; benefits and shortcomings of the methods are discussed.
Labeling AntibodiesEric A. Berg & Jordan B. Fishman, Cold Spring Harb Protoc., 2020 Jul 1;2020(7):099242.
This introduction outlines general strategies for labeling proteins, with an emphasis on methods that are used primarily for labeling antibodies. It covers the specific site of modification, cross-linker options, types of labels, and postlabeling cleanup methodology, along with the advantages and disadvantages of each method. In general, polyclonal antibodies are more versatile and resistant to activity loss than are monoclonal antibodies. Greater care must be taken when labeling monoclonal antibodies to ensure a quality conjugate. The methods outlined here can be adapted for a variety of labels including multiple labels on the same immunoglobulin. The most important consideration when undertaking an antibody labeling experiment is to maintain the activity of the antibody. This is an empirical process and will often require additional experiments to optimize the label of a particular antibody. When successful, these reagents are very useful and adaptable biomolecules. This introduction provides the reader with methods and options for producing a variety of labeled immunological tools.
Enzyme-based protein-tagging systems for site-specific labeling of proteins in living cellsShinji Sueda, Microscopy (Oxf), 2020 May 21;69(3):156-166.
Various protein-labeling methods based on the specific interactions between genetically encoded tags and synthetic probes have been proposed to complement fluorescent protein-based labeling. In particular, labeling methods based on enzyme reactions have been intensively developed by taking advantage of the highly specific interactions between enzymes and their substrates. In this approach, the peptides or proteins are genetically attached to the target proteins as a tag, and the various labels are then incorporated into the tags by enzyme reactions with the substrates carrying those labels. On the other hand, we have been developing an enzyme-based protein-labeling system distinct from the existing ones. In our system, the substrate protein is attached to the target proteins as a tag, and the labels are incorporated into the tag by post-translational modification with an enzyme carrying those labels followed by tight complexation between the enzyme and the substrate protein. In this review, I summarize the enzyme-based protein-labeling systems with a focus on several typical methods and then describe our labeling system based on tight complexation between the enzyme and the substrate protein.