Description
Selectivity and irreversible linkage stabilize crosslinked proteins Highly selective antibody labeling: reduces the probability of the label interfering with subsequent antibody-antigen reactions Irreversible linkage: stabilizes crosslinked proteins Provides an alternative when CyDye NHS esters are unsuitable: such as antibodies where amine labeling might impact antibody-antigen binding affinity Thiol groups are useful targets for protein labeling. Because they are present in most proteins, but less common than primary amines, crosslinking is more selective and precise. Crosslinking at these sites typically does not significantly modify the underlying protein structure or block binding sites. Maleimides react specifically with thiol groups such as cysteine residues in peptides, proteins, and oligonucleotides. This yields an irreversible thioether linkage, stabilizing crosslinked proteins for protein-protein interaction studies. Cy3 and Cy5 labeled maleimides are used to create fluor-labeled protein conjugates. They are often used in steady-state fluorescence, fluorescence polarization (FP) and fluorescence resonance energy transfer (FRET) applications to study protein-protein, protein-peptide, and protein-nucleic acid interactions. CyDye labeled maleimides are also used for antibody labeling since the dye molecules are introduced away from the antibody binding site. This increases the likelihood that functionality of the labeled molecule will be unimpaired. The protocol is simple and robust. This offers an alternative chemistry when either greater selectivity of labeling is required or amine labeling with Cy3 and Cy5 NHS esters is not desirable or appropriate. Each kit contains five vials of dye, each sufficient for labeling 1 mg of protein. Dyes are packaged in premeasured amounts and foil-sealed to ensure consistent labeling.
