posted on 2020-01-26, 18:29authored byShaheen Jeeawoody, Kevin A. Yamauchi, Alison Su, Amy E. Herr
Immunoprobed isoelectric
focusing (IEF) resolves proteins based
on differences in isoelectric point (pI) and then
identifies protein targets through immunoprobing of IEF-separated
proteins that have been immobilized onto a gel scaffold. During the
IEF stage, the gel functions as an anti-convective medium and not
as a molecular sieving matrix. During the immunoprobing stage, the
gel acts as an immobilization scaffold for IEF-focused proteins via
photoactive moieties. Here, we characterized the effect of gel pore
size on IEF separation and in-gel immunoassay performance. We modulated
polyacrylamide (PA) gel pore size via lateral chain aggregation initiated
by PEG monomers. During IEF, the 2% PEG highly porous PA gel formulation
offered higher resolution (minimum pI difference
∼0.07 ± 0.02) than unmodified 6%T, 3.3%C (benchmark) and
6%T, 8%C (negative control) PA gels. The highly porous gels supported
a pH gradient with slope and linearity comparable to benchmark gels.
The partition coefficient for antibodies into the highly porous gels
(K = 0.35 ± 0.02) was greater than the benchmark
(3×) and negative control (1.75×) gels. The highly porous
gels also had lower immunoassay background signal than the benchmark
(2×) and negative control (3×) gels. Taken together, lateral
aggregation creates PA gels that are suitable for both IEF and subsequent
in-gel immunoprobing by mitigating immunoprobe exclusion from the
gels while facilitating removal of unbound immunoprobe.