The BroadPharm NHS-(PEG)n is a amine-reactive reagent with an extended spacer arm. This reagent is soluble in organic solvents such as DMSO or DMF. Once dissolved in an organic solvent, the reagent is further diluted in a non-amine containing aqueous buffer. N-Hydroxysuccinimide (NHS) ester-activated PEG linker is an amine-reactive reagent. NHS esters react efficiently with primary amino groups (-NH2) in pH 7-9 buffers to form stable amide bonds. Because antibodies and other proteins generally contain multiple lysine (K) residues in addition to the N-terminus of each polypeptide, they have multiple primary amines available as targets for labeling with NHS-activated PEG reagents.
The NHS-(PEG)n is moisture-sensitive. Store the vial of the reagent at -20░C with desiccant. To avoid moisture condensation onto the product, equilibrate vial to room temperature before opening.
Dissolve the NHS-(PEG)n immediately before use. The NHS-ester moiety readily hydrolyzes and becomes non-reactive; therefore, weigh and dissolve only a small amount of the reagent at a time and do not prepare stock solutions for storage. Discard any unused reconstituted reagent.
Avoid buffers containing primary amines (e.g. Tris or glycine) as these compete with the intended reaction. If necessary, dialyze or otherwise desalt to exchange the protein sample into an amine-free buffer such as phosphate buffered saline.
Additional Materials Required
Additional Materials Required
Phosphate-buffered Saline (PBS): 0.1M phosphate, 0.15M sodium chloride; pH 7.2 or other non-amine containing buffer at pH 7.0-8.0
uenching Buffer: Tris-buffered saline (TBS; 25mM Tris, 0.15M sodium chloride; pH 7.2; glycine or other amine-containing buffer)
Water-miscible organic solvent such as dimethylsulfoxide (DMSO) or dimethylformamide (DMF)
10-100 ÁL sample volumes; Slide-A-Lyzer« Dialysis Cassette Kit for 0.1-30.0 mL sample volumes; or Zeba Spin Desalting Columns for sample volumes ranging from >10 ÁL to 4 mL
Procedure for labeling IgG with NHS-(PEG)n
The extent of PEG linker labeling depends on the size and distribution of amino groups on the protein and the amount of reagent used. Compared to reactions involving concentrated protein solutions, labeling reactions with dilute protein solutions require a greater fold molar excess of the NHS-(PEG)n linker to achieve the same incorporation level. Typically using a 20-fold molar excess of the NHS-(PEG)n linker to label 1-10 mg/mL antibody (IgG) results in 4-6 linkers labeling per antibody molecule. Adjust the molar ratio of NHS-(PEG)n to protein to obtain the desired level of incorporation.
Calculate millimoles of NHS-(PEG)n to add to the reaction for a 20-fold molar excess.
Calculate microliters of 10mM NHS-(PEG)n preparation for adding to the reaction.
B. NHS-(PEG)n Labeling Reaction
For reaction volumes from 10 ÁL to 100 ÁL, the buffer exchange and pegylation may be conveniently performed in a single Slide-A-Lyzer MINI Dialysis Unit. For reaction volumes from 0.1 mL to 30 mL, Slide-A-Lyzer Dialysis Cassettes may be used. Alternatively, Zeba Spin Desalting Columns can be used for a faster buffer exchange.
Equilibrate the vial of NHS-(PEG)n to room temperature before opening in Step 3.
Dissolve 1-10 mg protein in 0.5-2 mL of PBS according to the calculation made.
Immediately before use, prepare a 10mM solution of NHS-(PEG)n by adding about 5 mg into 1 mL of DMSO or DMF.
Add the appropriate volume of the NHS-(PEG)n solution (a 20-fold molar excess) to the protein solution, making sure that the volume of organic solvent does not exceed 10% of the final reaction volume.
Incubate reaction on ice for two hours or at room temperature for 30-60 minutes.
Remove the unreacted NHS-(PEG)n by dialysis or gel filtration. See instructions provided with the preferred buffer exchange product.
Store the pegylated protein using the same condition that is optimal for the non-pegylated protein.
The PEG NHS esters provide efficient PEGylation with primary amines at pH 7-9.
Reaction of the NHS-ester group results in the formation of a stable, irreversible