Borenpharm BIS PFP (PEG)n Reagents are homobifunctional crosslinkers for covalent conjugation between amine-containing molecules. Crosslinkers having polyethylene glycol (PEG) spacers are convenient and useful alternatives to those with purely hydrocarbon spacer arms. PEG spacers improve water-solubility of reagent and conjugate, reduce the tendency of conjugates to aggregate upon storage, and decrease immunogenic response to the spacer itself. By contrast to typical PEG reagents that contain heterogeneous mixtures of different PEG chain lengths, Pierce PEG reagents are homogeneous compounds of defined molecular weight and spacer arm length, providing greater precision in optimization and characterization of crosslinking applications. Homobifunctional crosslinkers containing pentaflourophenyl (PFP) esters are often used for low-resolution 3-D studies of protein structure and protein interaction analysis.
Accessible α-amine groups at the N-termini of proteins and peptides and the ε-amine of lysine residues react with PFP esters at pH 7-9 to form covalent amide bonds. The reaction results in the release of PFP. Hydrolysis of the PFP ester is the major competing reaction, the rate of which increases with pH and occurs more readily in dilute protein solutions. PFP ester crosslinking reactions are most commonly performed in phosphate, carbonate/bicarbonate, HEPES and borate buffers. Other buffers may also be used, provided they do not contain primary amines such as Tris or glycine. Using a large excess of Tris or glycine at neutral-to-basic pH can quench the reaction.
Important Product Information
BIS PFP (PEG)n Reagents are viscous pale liquids that are difficult to weigh and dispense. To facilitate handling, make a stock solution immediately before first use by dissolving the crosslinker in dry (anhydrous, molecular sieve-treated) organic solvent, such as dimethylsulfoxide. Minimize reagent exposure to moisture because the PFP-ester reactive group is susceptible to hydrolysis. Store unused stock solution in a moisture-free condition (e.g., capped under an inert gas such as argon or nitrogen) at -20°C. Equilibrate reagent vial to room temperature before opening to avoid moisture condensation inside the container. Minimize exposure to air by keeping the stock solution capped by a septum through which reagent can be obtained with a syringe. With proper handling, the stock solution is stable for three months.
Avoid buffers containing primary amines (e.g., Tris or glycine) during conjugation because they will compete with the intended reaction. If necessary, dialyze or desalt samples into a buffer such as phosphate-buffered saline (PBS).
The crosslinker-to-protein molar ratio affects the modification extent of available amine groups and, therefore, crosslinking. This ratio requires optimization to yield the extent of crosslinking best for the specific application.