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I observe a rather unusal binding result. Increasing the
concentration of GST-fusion protein over quite a wide range (5-fold
concentration difference-2, 4, 6, 8, 10 ml of bacterial cell lysate
that resulted in a linear increase in purified protein from 6.3 ug
for the 2 ml sample to 24.8 ug for the 10 ml sample) results in a
near linear increase in protein binding to glutathione beads (20 ul
of MagneGST beads). My assumption was that I did not have excess GST-
fusion protein present and therefore was not saturating the beads.
However, in the same experiment, to the 10 ml bacterial cell lysate
sample I added 100 ul of MagneGST beads thinking I would only be able
to purify around 25 ug of GST-fusion protein, but I actually purified
60 ug or roughly 2.4-fold more than what I purifed using 20 ul of
beads. Clearly there was a large excess of unbound GSt-fusion protein
present in the sample with 20 ul of beads, so why did the amount
purified increase linearly from 2 ml to 10 ml of lysate?
Clearly in the 8 ml sample there was also a large excess of GST-
fusion protein available for binding, yet rather than observe the
saturation of the beads they amount bound just keeps going up
linearly. The puriity of all samples is similar as well so the
additional protein eluted is not just non-specifically bound protein.
Any advice as to what is going on? My thinking is that the binding is
non-equilibrium, but really I do not see any difference between 10
min of binding and 30 min of binding at the same bead and sample
amounts suggesting that maybe there are 2 bidning states--one fast
and one slow and maybe with a huge excess of beads you can capture
some of the "slow" binding but at low bead amounts you can't?
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