PEG NHS Ester is an amine-reactive reagent with a PEG spacer of specified length. It dissolves in organic solvents like DMSO or DMF and then dilutes in amine-free aqueous buffers. PEG NHS Ester efficiently reacts with primary amines (-NH2) in neutral or slightly basic buffers, forming stable amide bonds. Antibodies and proteins, rich in lysine residues and N-terminal amines, serve as ideal targets for labeling with NHS-activated PEG reagents, enhancing their functionality and solubility.
Condition 1: Base (TEA, DIPEA, Py, etc.); DCM or DMF
Condition 2: PBS buffer, pH 7.4-9.
Product Information
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Moisture Sensitivity: PEG NHS Ester is highly sensitive to moisture. Store it at -20°C with a desiccant. Let the vial warm to room temperature before opening to avoid moisture condensation.
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Preparation: Dissolve PEG NHS Ester immediately before use. The NHS-ester group hydrolyzes quickly, losing reactivity. Weigh and dissolve only small amounts as needed, and avoid making stock solutions. Discard any unused dissolved reagent.
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Buffer Compatibility: Avoid buffers containing primary amines (e.g., Tris or glycine) as they will interfere with the reaction. If needed, dialyze or desalt the protein sample to an amine-free buffer, such as phosphate-buffered saline (PBS).
Additional Materials Required
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Phosphate-Buffered Saline (PBS): Use PBS with 0.1M phosphate and 0.15M sodium chloride at pH 7.2, or select another non-amine buffer with a pH between 7.0 and 8.0.
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Quenching Buffer: Use Tris-buffered saline (TBS) with 25mM Tris and 0.15M sodium chloride at pH 7.2, or glycine and other amine-containing buffers.
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Water-Miscible Organic Solvent: Choose from dimethyl sulfoxide (DMSO) or dimethylformamide (DMF).
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Sample Handling: For 10-100µL sample volumes, use the Slide-A-Lyzer® Dialysis Cassette Kit for 0.1-30.0mL or Zeba Spin Desalting Columns for volumes >10µL to 4mL.
Procedure for Modifying Amine-Bearing Small Molecules with PEG NHS Ester
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Dissolve Small Molecules: Gradually dissolve the amine-bearing molecules in an anhydrous organic solvent like DMF, DCM, DMSO, or THF. Choose a solvent suitable for your specific substrate.
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Add Base and PEG NHS Ester: While stirring, add a base and PEG NHS Ester to the reaction mixture. Use a 1:1 or 2:1 molar ratio based on the reaction kinetics.
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Stir and Monitor: Stir the reaction mixture continuously for 3-24 hours, adjusting the time based on the substrate properties. Monitor the reaction progress using LC-MS or TLC.
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Isolate the Product: Isolate the final product using standard organic synthesis workup methods or column purification.
Procedure for Labeling IgG with PEG NHS Ester
A. Calculations
- Determine PEG labeling based on the number and distribution of amino groups on the protein and the amount of reagent used.
- Dilute protein solutions require a higher molar excess of PEG NHS Ester than concentrated ones to achieve similar labeling.
- Typically, a 20-fold molar excess of PEG NHS Ester labels 1-10 mg/mL of IgG, resulting in 4-6 linkers per antibody.
- Adjust the NHS-(PEG)n to protein ratio to achieve the desired level of incorporation.
Steps:
- Calculate the required millimoles of PEG NHS Ester for a 20-fold molar excess.
- Determine the microliters of a 10mM PEG NHS Ester solution needed for the reaction.
B. PEG NHS Ester Labeling Reaction
- For 10µL to 100µL volumes, perform buffer exchange and PEGylation in a Slide-A-Lyzer MINI Dialysis Unit.
- For 0.1mL to 30mL, use Slide-A-Lyzer Dialysis Cassettes. Use Zeba Spin Desalting Columns for quicker buffer exchange if needed.
Steps:
- Let PEG NHS Ester reach room temperature before opening.
- Dissolve 1-10 mg of protein in 0.5-2 mL PBS based on calculated needs.
- Prepare a 10mM PEG NHS Ester solution by dissolving ~5 mg in 1 mL DMSO or DMF just before use.
- Add the PEG NHS Ester solution (20-fold excess) to the protein solution. Ensure the organic solvent is less than 10% of the final volume.
- Incubate on ice for 2 hours or at room temperature for 30-60 minutes.
- Remove unreacted PEG NHS Ester by dialysis or gel filtration.
- Store the PEGylated protein under the same conditions as the original protein.