PEG NHS Ester is a type of amine-reactive reagent that features a PEG spacer of a specified length. This reagent is soluble in organic solvents like DMSO or DMF. After dissolving in an organic solvent, it is further diluted in an aqueous buffer that does not contain amines. PEG NHS Ester reacts efficiently with primary amino groups (-NH2) in neutral or slightly basic buffers to form stable amide bonds. Since antibodies and other proteins typically have multiple lysine (K) residues in addition to the N-terminus of each polypeptide, they offer several primary amines as targets for labeling with NHS-activated PEG reagents.
Condition 1: Base (TEA, DIPEA, Py, etc.); DCM or DMF
Condition 2: PBS buffer, pH 7.4-9.
Product Information
• PEG NHS Ester is sensitive to moisture. Store the reagent vial at -20°C with a desiccant. To prevent moisture condensation on the product, allow the vial to reach room temperature before opening.
• Dissolve PEG NHS Ester just before use. The NHS-ester group hydrolyzes quickly and loses reactivity, so only weigh and dissolve a small amount at a time and avoid making stock solutions for storage. Discard any unused dissolved reagent.
• Avoid using buffers that contain primary amines (e.g., Tris or glycine) as they will interfere with the intended reaction. If necessary, dialyze or desalt the protein sample to switch it to an amine-free buffer such as phosphate-buffered saline.
Additional Materials Required
• Phosphate-buffered Saline (PBS): Contains 0.1M phosphate and 0.15M sodium chloride, with a pH of 7.2, or use another non-amine buffer with a pH between 7.0 and 8.0.
• Quenching Buffer: Tris-buffered saline (TBS) with 25mM Tris and 0.15M sodium chloride, pH 7.2; or use glycine or other amine-containing buffers.
• Water-miscible organic solvent: Options include dimethyl sulfoxide (DMSO) or dimethylformamide (DMF).
• 10-100µL sample volumes; Slide-A-Lyzer® Dialysis Cassette Kit for 0.1-30.0mL sample volumes; or Zeba Spin Desalting Columns for sample volumes ranging from >10µL to 4mL
Procedure for Modifying Amine-Bearing Small Molecules with PEG NHS Ester
Gradually dissolve the amine-bearing small molecules in anhydrous organic solvents such as DMF, CH2Cl2, DMSO, THF, or other suitable solvents. While continuously stirring, add a base and PEG NHS Ester to the reaction mixture in a 1:1 or 2:1 molar ratio, depending on the reaction kinetics. Stir the reaction mixture for 3-24 hours, depending on the properties of the substrate, and monitor the progress using LC-MS or TLC. The final product can be isolated through standard organic synthesis workup procedures or column purification.
Procedure for Labeling IgG with PEG NHS Ester
A. Calculations
The degree of PEG linker labeling depends on the size and distribution of amino groups on the protein and the amount of reagent used. Labeling reactions with dilute protein solutions require a higher molar excess of the PEG NHS Ester linker compared to reactions with concentrated protein solutions to achieve the same level of incorporation. Typically, using a 20-fold molar excess of the PEG NHS Ester linker to label 1-10 mg/mL of antibody (IgG) results in 4-6 linkers per antibody molecule. Adjust the molar ratio of NHS-(PEG)n to protein to achieve the desired level of incorporation.
- Calculate the millimoles of PEG NHS Ester needed for a 20-fold molar excess.
- Calculate the microliters of a 10mM PEG NHS Ester solution to add to the reaction.
B. PEG NHS Ester Labeling Reaction
For reaction volumes between 10µL and 100µL, buffer exchange and PEGylation can be conveniently performed in a single Slide-A-Lyzer MINI Dialysis Unit. For reaction volumes from 0.1mL to 30mL, use Slide-A-Lyzer Dialysis Cassettes. Alternatively, Zeba Spin Desalting Columns can be used for faster buffer exchange.
- Allow the vial of PEG NHS Ester to reach room temperature before opening in Step 3.
- Dissolve 1-10 mg of protein in 0.5-2 mL of PBS based on the calculated amount.
- Just before use, prepare a 10mM solution of PEG NHS Ester by dissolving approximately 5 mg in 1 mL of DMSO or DMF.
- Add the appropriate volume of the PEG NHS Ester solution (a 20-fold molar excess) to the protein solution, ensuring that the volume of organic solvent does not exceed 10% of the final reaction volume.
- Incubate the reaction on ice for two hours or at room temperature for 30-60 minutes.
- Remove any unreacted PEG NHS Ester by dialysis or gel filtration.
- Store the PEGylated protein under the same conditions that are optimal for the non-PEGylated protein.