Introduction
The PFP Ester is a class of PEG linker reagents that react with primary and secondary amines to form amide bonds. The pentafluorophenyl (PFP) ester is less subject to hydrolysis than NHS esters, resulting in more efficient reactions.
Materials & Reagents:
· PFP ester-activated linker
· Biomolecule with a free amine (e.g., protein, peptide, or amine-modified RNA)
· Reaction buffer: 50–100 mM PBS, borate, carbonate/bicarbonate, HEPES buffer, pH 7.2–8.5
· Organic co-solvent (if needed): DMSO, DMF, ACN
· Mild base (if needed): 0.1 M NaHCO₃ or 10–50 mM triethylamine (TEA)
· Quenching reagent (optional): Tris buffer (if excess PFP ester needs to be deactivated)
Procedure:
1. Prepare the Biomolecule Solution
1) Dissolve the biomolecule in 50–100 mM reaction buffer, pH 7.2–8.5.
2) If the biomolecule is prone to aggregation, add 5–10% DMSO or DMF to improve solubility.
3) Keep the final biomolecule concentration between 0.5–5 mg/mL, depending on the application.
2. Prepare the PFP Ester Solution
1) Dissolve the PFP ester-activated linker in DMSO, DMF, or ACN at a 10–100 mM concentration.
2) If needed, dilute this stock with reaction buffer immediately before use.
3. Initiate the Conjugation Reaction
1) Add the PFP ester solution slowly to the biomolecule solution while stirring.
2) Maintain a molar ratio of PFP ester to free amine between 2:1 and 10:1 (optimize based on efficiency).
3) Allow the reaction to proceed at room temperature (20–25°C) for 1–4 hours or at 4°C overnight for sensitive biomolecules.
4) Optional: If the reaction is slow, add a mild base (e.g., 10–50 mM TEA or 0.1 M NaHCO₃) to improve amine reactivity.
4. Monitor Reaction Progress
· If the biomolecule is a protein or peptide, confirm conjugation via: HPLC or LC-MS (to check molecular weight shift)
· SDS-PAGE (for size changes in proteins)
If working with small molecules or DNA, use LC-MS or MALDI-TOF for analysis.
5. Quenching Excess PFP Ester (If needed)
· If excess PFP ester needs to be removed, add Tris buffer (pH 8.0–8.5) and incubate for 30 minutes.
6. Purification of Conjugated Biomolecule
· Desalting Columns (e.g., Sephadex G-25, PD-10) for proteins/peptides
· Dialysis (if buffer exchange is required)
· HPLC or SEC Purification (for small molecules or precise fractionation)
· Precipitation (DNA or RNA could precipitate in acetone)
Notes & Optimization Tips:
· Reaction pH: pH 7.2–8.5 is optimal; lower pH reduces reactivity, while higher pH may cause hydrolysis.
· Solvent Choice: If solubility is an issue, mix 5–10% DMSO, DMF or ACN in the buffer.
· Reaction Efficiency: Optimize ester-to-amine ratio and time for better yield.