PEG-NHS Ester Compounds

Most of these PEG crosslinkers come as thick, sticky, viscous liquids or low melting solids (PEG2). For this reason, weighing out a small sample of a compound can be difficult or impossible. It usually is best to dissolve an entire vial or a larger amount in organic solvent at a known concentration to permit accurate dispensing of a smaller amount into a reaction. Suitable solvents to prepare a stock solution include dry (molecular sieved) DMSO, DMF, DMAC, acetonitrile, or methylene chloride (for non-water-miscible reactions). 

In a fume hood, dissolve the NHS-PEG -maleimide compound of choice in a dry, water-miscible organic solvent to make a concentrated stock solution. For instance, to prepare a lOOmM solution of the NHS-PEGg-maleimide reagent (MW 601.6), dissolve an entire 100 mg vial of the crosslinker in 1.66 ml of DMAC (dry DMSO or DMF work well, too). 

Dissolve the amine-containing protein to be activated in 0.1 M sodium phosphate, pH 7.2 (coupling buffer). A protein concentration of l-10mg/ml in buffer will work well in this protocol. For more dilute protein solutions, greater quantities of the NHS-PEG,-maleimide compound may have to be added to obtain equivalent levels of modification. 

Add a quantity of the crosslinker solution to the protein solution to provide at least a 10-fold molar excess of reagent over the amount of protein present. Higher levels of reagent-to-protein may be used, even up to a 50-fold molar excess, to obtain a large number of active groups for subsequent coupling to a thiol-protein. Conversely, the use of lower molar ratios will limit the number of maleimide-PEG modifications on each 

Protein modified via amide bond and having terminal maleimide available for coupling with second protein 

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In addition, the PEG-based heterobifunctional crosslinkers described in Chapter 18, Section 2, provide enhanced water-solubility for antibody conjugation applications. Conjugation of antibody molecules using a maleimide-PEG -NHS ester compound actually increases the solubility of the antibody and may help to maintain stability for certain sensitive monoclonals better than the traditional aliphatic crosslinkers. The methods described below for SMCC may be used with success for PEG-based reagents or other maleimide-NHS ester heterobifunctionals. 

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