Application Instructions for Y-NHS-40K for Amine PEGylation

We describe a general protocol for protein PEGylation using our proprietary Y-shape PEG NHS ester, Y-NHS-40K. Proteins typically contain lysines with primary amines that can be PEGylated by Y-shape PEG NHS esters. In pH 7-7.5 buffers, Y-shape PEG NHS esters react efficiently with primary amino groups on the side chain of lysines by nucleophilic attack forming amide bonds and releasing free NHS.


The process of PEGylation, the covalent conjugation of a PEG derivative onto molecules, improves the water solubility and biocompatibility of those molecules, a beneficial feature for protein drug development [1]. The sterically bulky structure of JenKem Technology’s proprietary Y-shape branched PEG derivatives, consisting of two linear methoxy PEG chains attached to a central active core, may help to reduce the number of attachment sites to a protein molecule. As a PEG NHS ester, Y-shape PEG NHS ester is readily dissolved in aqueous buffers and enables simple and efficient modification of proteins and other biological agents that contain lysines.

Compared with linear PEG NHS esters, under typical PEGylation conditions, JenKem Technology’s Y-shape PEG NHS esters have increased selectivity towards more sterically available amines, due to their bulky structure [2-6]. Protein PEGylation with Y-shape PEG NHS esters can be accomplished in less than one hour under the suggested reaction conditions.


Y-NHS-40K (Y-shape PEG NHS Ester, MW 40000, JenKem Technology item code A0001)

Phosphate buffer or other amine-free buffer of your choice at pH 7.0-7.5

Protein to be PEGylated

Dry water-miscible solvent (e.g., dry DMF or DMSO)

Figure 1. Chemical structure of Y-NHS-40K (Y-shape PEG NHS Ester, MW 40000, JenKem Technology item code A0001)

Y-shape PEG NHS Ester




Protocol for Protein PEGylation with Y-NHS-40K

1. Sample Storage and Handling

All PEG derivatives should be stored under a dry inert gas (argon or nitrogen), in the dark, at or below -20°C.

In addition, PEG succinimidyl active esters, including Y-NHS-40K, will hydrolyze in water. It is essential to minimize exposure of these products to moisture and to keep them cold to reduce the degree of hydrolysis. JenKem Technology’s PEG derivatives are thoroughly dried and sealed under dry nitrogen/argon before shipping. JenKem Technology’s PEG derivatives should be stored desiccated at or below -20°C upon receiving.

Before using PEG derivative after storage, allow the bottle containing the reagent to warm to room temperature slowly. After using, the reagent container should be backfilled with a dry inert gas (argon or nitrogen) and store again at or below -20°C. For multiple uses, in order to avoid repeated cycling through multiple freeze-thaw cycles, it is a good practice to divide PEG derivatives into several small portions at the first use. You can also request repacking service when you place the order.

2. PEGylation Protocol using JenKem Technology’s Y-NHS-40K

We describe here a general 5 step-protocol for initial studies of protein PEGylation using JenKem Technology’s Y-shape PEG NHS ester (Y-NHS-40K). As mentioned in the introduction, Y-NHS-40K reacts efficiently with primary amino groups on the side chain of lysines on protein at pH 7-7.5, forming amide bonds, along with releasing free NHS.

Figure 2. Schematic of protein PEGylation using JenKem Technology’s Y-NHS-40K

Y-NHS PEGylation

Step 1: 

Take the bottle containing Y-NHS-40K from storage and fully equilibrate it to room temperature before use. Remember to seal under inert gas (argon or nitrogen), and keep at or below -20°C after use.

Step 2:

Calculate the amount (mole or mmole) of protein to be PEGylated and dissolve the protein in a phosphate buffer or other amine-free buffer of your choice at pH 7.0-7.5. 

Proteins stored in Tris or other amine-containing buffers must be exchanged into a suitable buffer before use.

Step 3:

Depending on the nature of the protein and the degree of PEGylation desired, the amount of Y-NHS-40K to be used for each PEGylation process differs. As a starting point, consider using a 5- to 10- folds molar excess of Y-PEG-NHS for protein solutions concentration of not less than 2 mg/mL. When protein solution concentration is more dilute, a greater relative molar excess of Y-NHS-40K may be necessary for desired PEGylation results.

Calculate the amount of Y-NHS-40K to be used in PEGylation reaction, dissolve the Y-NHS-40K in dry water-miscible solvent (e.g., dry DMF or DMSO) and add slowly to the protein solution with a gentle swirl.

Step 4:

Incubate reaction mixture at 0-5ºC for about three hours, or at room temperature for about one hour.

Reaction time may vary with the nature of proteins. Unless for possible protein degradation or microbial growth concerns, there should be no harm in keeping the reaction mixture for longer at above conditions.

Step 5:

When PEGylation reaction is finished, the degree of PEGylation should be evaluated. Mono PEGylated protein can be purified from multiple-PEGylated protein and non-PEGylated protein through chosen chromatographic methods.


We provided general application instructions for amine PEGylation of proteins with Y-NHS-40K, useful for initial protein PEGylation studies. PEGylation of proteins with Y-shape PEG NHS esters can be achieved in less than an hour at room temperature in pH 7-7.5 buffer.


JenKem Technology may provide technical assistance and information with respect to JenKem Technology’s PEG derivatives ordered by our customers. Any and all such technical assistance shall be on “as-is” basis, and JenKem Technology makes no warranties of any kind or nature, express or implied, including without limitation, any implied warranty of merchant ability of fitness for any particular purpose or non-infringement with respect to technical assistance for information provided by JenKem Technology or JenKem Technology’s personnel. Any suggestions by JenKem Technology regarding use, selection, application or suitability of the PEG derivatives shall not be construed as an express or implied warranty unless specifically designated as such in a writing signed by an officer of JenKem Technology.


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  2. Roccaro, Aldo M. et al., SDF-1 Inhibition Targets the Bone Marrow Niche for Cancer Therapy, Cell Reports , 9 (1), 2014.p: 118 – 128.
  3. Ashokan, A., et al., Multifunctional calcium phosphate nano-contrast agent for combined nuclear, magnetic and near-infrared in vivo imaging. Biomaterials, 2013. 34(29): p. 7143-7157.
  4. Khan, M.A., et al., Targeting complement component 5a promotes vascular integrity and limits airway remodeling, PNAS 2013 110(15) p:6061-6066.
  5. Dai, C.Y., et al., Linkage with cathepsin B-sensitive dipeptide promotes the in vitro and in vivo anticancer activity of PEGylated tumor necrosis factor-alpha (TNF-α) against murine fibrosarcoma, Science China Life Sci 2011 54(2): 128–138.
  6. Menkhorst, E., et al., Vaginally Administered PEGylated LIF Antagonist Blocked Embryo Implantation and Eliminated NonTarget Effects on Bone in Mice, PLoS ONE, 2011, 6 (5) e19665.