Activated PEGs for PEGylation

　1) PEG-conjugate

Following figure shows several examples of PEG-aldehyde conjugation with human insulin for various molecular weights of PEG.


Analysis of PEG-ALD-Insulin by SDS-PAGE with Coomassie brilliant blue.
Insulin was reacted with NOF PEG-ALD at 4℃. The PEGylaed insulin was purified on Q Sepharose Fast Flowcolumn.

lane M molecular weight markers	lane 3 PEG(ME-100AL)-insulin eluate
lane 1 insulin	lane 4 PEG(ME-200AL)-insulin eluate
lane 2 PEG(ME-050AL)-insulin eluate	lane 5 PEG(ME-300AL)-insulin eluate

　 2) Coupling of PEG derivatives to Proteins: examples of conditions

1. Coupling of PEG-NHS derivatives to protein amines (PEG-NHS + Protein-NH₂) Conditions 1: 50 mM phosphate buffer (pH 7.2), 4℃, 6 hr^*1)
   Conditions 2: Borate-phosphate buffer (pH 8.0), 25℃, 2 hr^*2)
2. Coupling of PEG-Aldehyde derivatives to the NH₂ group of proteins (PEG-Ald + Protein-NH₂)
   Conditions : sodium cyanoborohydride (10 equiv.), 4℃, 20 hr^*3)
3. Coupling of PEG-Maleimide derivatives to the SH group of proteins (PEG-Maleimide + Protein-SH)
   Conditions: 100 mM phosphate buffer (pH 6.5), 4℃, 4 hr
4. Coupling of PEG-NH₂ derivatives to the COOH group of proteins (PEG-NH₂ + Protein-COOH)
   Conditions: 50mM phosphate buffer (pH 7.2), WSC (2 equiv.), 4℃, 10 hr
5. Coupling of PEG-p-Nitrophenyloxycarbonyl derivatives to the NH₂ group of proteins (PEG-NP + Protein-NH₂)
   Conditions: borate-phosphate buffer (pH 8.0-8.3), rt, overnight^*2)

References:
*1) Abuchowski, A., et al. (1984) Cancer Biochem. Biophys. 7, 175.
*2) Sartore, L., et al. (1991) Appl. Biochem. Biotechnol. 27, 45
*3) US 5,824,784