Proteins must maintain
proper folding conformations and express
the correct post-translational modifications (PTMs) to exhibit appropriate
biological activity. However, assessing protein folding and PTMs is
difficult because routine polyacrylamide gel electrophoresis (PAGE)
methods lack the separation resolution necessary to identify variants
of a single protein. Additionally, standard PAGE denatures proteins
prior to analysis precluding determinations of folding states or PTMs.
To overcome these limitations, a microfluidic thermal gel electrophoresis
platform was developed to provide high-sensitivity, high-resolution
analyses of native protein variants. A thermally reversible gel was
utilized as a separation matrix while in its solid state (30 °C).
This thermal gel provided sufficient separation resolution to identify
three variants of a fluorescently labeled model protein. To increase
detection sensitivity, analyte preconcentration was conducted in parallel
with the separation. Continuous analyte enrichment afforded detection
limits of 500 fg of protein (250 pM) while simultaneous baseline separation
resolution was achieved between variants. The effects of temperature
on thermal gel electrophoresis were also characterized. The unique
temperature-dependent outcomes illustrated how method performance
can be tuned through a thermal dimension. Ultimately, the high detection
sensitivity and separation resolution provided by thermal gel electrophoresis
enabled rapid screening of native protein variants.
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