I've conjugated a PEG4-maleimide (MW 613.66) onto an antibody fragment (52 kDa), but I'm trying to figure out how this affects A260/280 measurements on a Nanodrop afterwards. After conjugation and removal of unconjugated material on a PD-10 column, I measure the concentration of the eluted aliquots but the measurements seem really off, and the A260/A280 ratio is nowhere close to 0.5. I blanked the Nanodrop with the PD-10 elution buffer. Seems strange that such a small molecule could affect the readings that much. Does anyone have experience with PEG and if/how it could affect concentration measurements after conjugation?
Did you equilibrate your PD-10 column sufficiently? Which buffer did you use?
Yes I equilibrate it with at least 25 mL of buffer: 0.1M HEPES, 0.5M NaCl, 10% glycerol, pH 7.
It is difficult to say, whether the PEG-maleimide derivative has a significant absorbance in this range after conjugation, as long as spectra are not available. The PEG group should not be critical, but the maleimide residue. I would suggest a BCA protein test, which is quite reliable in many cases.
Yes, I may need to run a BCA. Only problem is the upper limitation of 2 mg/mL for that assay but I suppose I could dilute my sample. Not sure how an ELISA would help determine protein concentration? Running a standard curve with SEC would probably work, but uses more sample than I'd like to.
Measurement of refractive index (RI, to which both PEG and protein contribute) and absorbance, e.g. at 280 nm or another wavelength during size-exclusion chromatography permits calculation of the separate contributions of the protein and the PEG to the absorbance and RI of the peak of the conjugate. See Kunitani et al., J. Chromatogr. 588:125-137 (1991).
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