Hello,
as a chemist, I'm currently working on the covalent conjugation of Maleimide-Neutravidin to furan-modified hyaluronic acid as precursor material for microbead preparation. To perform coupling of furan and maleimide via relatively slow Diels Alder click reaction I need to buffer the system with 0.1M MES-buffer pH 5.5 to prevent deactivation of maleimide funtionality. However, when I dissolve the lyophilized maleimide-neutravidin it forms aggregates. Is there any additive that I could add to aid dissolution at that pH? Does this pH somehow affect the followed Biotin-Neutravidin coupling?
Thanks for your help!
You have at least two problems here: At a pH of 5.5 you are approaching the isoelectric point of Neutravidin (6.3), which may lead to solubility issues. Normal avidin or streptavidin would be preferable. In addition, according to the manufacturer, the maleimide activation was performed with Sulfo-SMCC, a hydrophobic linker. This makes the conjugate even less soluble. I would prefer to use streptavidin activated with a low conjugation ratio of BMPS, which is smaller and is less hydrophobic.
Hi Thomas, I agree with Michael that this could be a solubility issue.
When reconstituting dry proteins there is a difference between protein aggregation and protein non-solubility.
Proteins are charged macromolecules that need to be hydrated for full solubility. Lyophilization removes the water so proteins stick to each other in the absence of a polar solvent.
Put a vial of your reconstituted protein into a simple sonicating waterbath. If this fixes the problem you are dealing with protein aggregates that need to be hydrated to get into solution.
If you still have aggregates, then you have a protein that is non-soluble and probably denatured.
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