The BroadPharm (PEG)n-Acid is a class of PEGs 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 witch containing amine groups such quantum dots, self-assembled monolayers and magnetic particles. Functionalization of solid surfaces with polyethylene glycol spacers significantly reduces nonspecific protein binding.
Storage : Upon receipt store desiccated at -20°C.
The (PEG)n-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).
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.
If the carboxyl-PEG reagent 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.
Use non-amine-containing buffers at pH 7-9 such as PBS (20mM sodium phosphate, 150mM NaCl; pH 7.4); 100mM carbonate/biocarbonate; or 50mM borate. Do not use buffers that contain primary amines, such as Tris or glycine, which compete with acylation.
Additional Materials Required
Water-miscible organic solvent (molecular sieve-treated) such as DMSO or DMF
Small-volume, non-coring syringes for dispensing the reagent stock solution while minimizing exposure to air
Buffer A: Phosphate-buffered saline, PBS (20mM sodium phosphate, 0.15MNaCl; pH 7.2) or other non-amine, lone-pair sulfur-free buffers
Buffer B: MES-buffered saline (0.1M MES, 0.5M NaCl; pH 6.0 or 0.1M MES, 0.9% NaCl; pH 4.7) or other non-amine, non-carboxy, lone-pair sulfur-free buffers
Equilibrate the PEG)n-Acid reagents to room temperature before opening bottles.
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 in the Important Product Information Section.
Prepare the appropriate amount of surface or protein in Buffer A.
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.
Add the desired amine-containing substrate, prepared in Buffer A, 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 Buffer A immediately before adding the amine-containing substrate.
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.
Prime, K.L. and Whitesides, G.M. (1991). Self-assembled organic monolayers: model systems for studying absorption of proteins at surfaces. Science 252:1164.
Bentzen, E.L., et al. (2005). Surface modification to reduce non-specific binding of quantum dots in live cell assays.BioconjugateChem16:1488-94.
Lin, P-C., et al. (2006). Ethylene glycol-protected magnetic nanoparticles for a multiplexed immunoassay in human plasma.Small2(4):485-9.
Zheng, M., et al. (2003). Ethylene glycol monolayer protected nanoparticles for eliminating nonspecific binding with biological molecules. J Am ChemSoc125:7790-1.
Verma, A. and Rotello, V.M. (2005). Surface recognition of biomacromolecules using nanoparticle receptors.ChemCommun3:303-12.
Kidambi, S., et al. (2004). Selective depositions on polyelectrolyte multilayers: self-assembled monolayers of m-dPEG acid as molecular template. J Am ChemSoc126:4697-03.
PEG-acid will react with an amine-containing moiety in the presence of a coupling
reagent. It can also be converted to N-hydroxysuccinimide active ester widely used