General Conjugation Protocols of PEG linkers——PEG Acid
Polyethylene glycol (PEG) linker is a chemical sensing the carrier of polyethylene glycol (PEG), due to its solubility in water and non-immunogenicity. In the field of scientific research, it is widely used in chemical coupling, drug delivery, nanoparticles functionalized modifications and chemical biology. Its powerful function has attracted intense research interest. In the following, we will show the general conjugate reactions of nine common polyethylene glycol linkers one by one.
PEG Acid
Introduction
PEG acid is a class of PEG compound containing a carboxylic acid group on one end and with either a hydroxyl, azido, amino, maleimide or a triple bond on the other end. These reagents have defined molecular weights and spacer lengths and are used for modifying proteins or surfaces containing amine groups such quantum dots, self-assembled monolayers and magnetic particles. Functionalization of solid surfaces with PEG spacers significantly reduces nonspecific protein binding.
Condition: 1 a) EDC, NHS, DMF or DCM, b) R-NH2
Condition: 2 a) 0.5 mM MES buffer, pH 4.5, EDC, NHS; b) R-NH2
Product Information
Note: Upon receipt store desiccated at -20 °C.
1) Most PEG Acid reagents are low-melting solids that are difficult to weigh and dispense. To facilitate handling, make a stock solution by dissolving the reagent in dimethylsulfoxide (DMSO) or dimethylformamide (DMF).
2) Store unused stock solution at -20 °C. Equilibrate reagent vial to room temperature before opening to avoid moisture condensation. To minimize air exposure, keep the stock solution under an inert gas such as argon or nitrogen. Cap the stock solution with a septum and use a syringe to remove the solution.
3) If the PEG Acid is used for surface binding and further protein loading, the reagent-to-surface ratio in the reaction affect the number of carboxylic acid residues available for further modification. Optimize these ratios to obtain the modification level needed for the specific application.
4) Use non-amine-containing buffers at pH 7-9 such as PBS (20 mM sodium phosphate, 150 mM NaCl; pH 7.4); 100mM carbonate/bicarbonate; or 50 mM borate. Do not use buffers that contain primary amines, such as Tris or glycine, which compete with acylation.
Additional Materials Required
1) Water-miscible organic solvent (molecular sieve-treated) such as DMSO or DMF.
2) Small-volume, non-coring syringes for dispensing the reagent stock solution while minimizing exposure to air.
3) PBS Buffer Phosphate-buffered saline, PBS (20mM sodium phosphate, 0.15 M NaCl; pH 7.2) or other non-amine, lonepair sulfur-free buffers.
4) MES Buffer: MES-buffered saline (0.1 M MES, 0.5 M NaCl; pH 6.0 or 0.1 M MES, 0.9% NaCl; pH 4.7).
5) EDC•HCl
6) NHS
7) Hydroxylamine•HCl
Procedure (organic solvent method)
PEG acid (1 mmol) was dissolved into dry DCM (1.5 mL). EDC•HCl’ (2.0 mmol) dissolved by 0.5 mL dry DCM and NHS (2.0 mmol) dissolved by 10 μL DMSO were added in sequence. The solution was stirred at room temperature for 30 min, and subsequently NH2-R’ (1.5 mmol) was dissolved into the solution. DIPEA (1.5 mmol) was added and the mixture stirred at room temperature for 1 h. the reaction can be separated by flash R18 to yield pure compound.
Procedure (aqueous method)
1) Equilibrate the PEG Acid reagents to room temperature before opening bottles.
2) Prepare stock solutions by dissolving 100 mg of each reagent in the desired amount of DMF or DMSO. Cap, store and handle stock solutions as directed amount of DMF or DMSO. Cap, store and handle stock solutions as directed in the Important Product Information Section.
3) Prepare the appropriate amount of surface or protein in PBS buffer.
4) The carboxylic acid groups on the PEG linker can be activated by adding appropriate amounts of EDC and NHS to the modified surface in a small amount of Buffer B and reacting for 15 minutes at room temperature. For best results, perform this reaction at pH 5-6. Note: The activation reaction with EDC and NHS is most efficient at pH 4.5-7.2; however, the reaction of NHS-activated molecules with primary amines is most efficient at pH 7-8.
5) Add the desired amine-containing substrate, prepared in PBS Buffer, to the activated surface and react for 2 hours at room temperature. For best results, raise the pH of the reaction solution to 7.2-7.5 with PBS buffer immediately before adding the amine-containing substrate.
6) To quench the conjugation reaction, add hydroxylamine or another amine-containing buffer. Hydroxylamine hydrolyzes non-reacted NHS. Other quenching compounds include Tris, lysine, glycine or ethanolamine; however, these primary amine-containing compounds modify carboxylic acids.