Hi Alex,
the binding of Coomassie Brilliant Blue R to proteins roughly correlates
with the number of positive charges (about 1.5-3 dye molecules/charge;
JBC 216, 9976-9980) whereas the SDS is thought to bind/align to the
protein backbone during/after heat denaturation (roughly 1 SDS per 2
amino acids). Because of the correlation to charge, the intensity of a
band on a gel is not a good indicator of protein concentration at all.
One or the other odd protein binds the dye so poorly that a negative
glass-clear band as compared to the slightly blue background of the gel
after destaining can be observed.
SDS is not fully wrapping/enveloping the unfolded protein, by binding to
the backbone it just prevents the refolding and by binding in a relative
stable ratio to the protein backbone (roughly 1 SDS per 2 amino acids),
induces a relative stable mass-charge distribution (a property that
nucleic acids own by nature) making the separation on a molecular sieve
solely based on mass possible in the first place. However, very basic,
acidic or post-translationally modified proteins will run differently on
SDS-PAGE than your average protein (and marker proteins).
Jeroen
Am 05.10.18 um 03:24 schrieb Alex Lee:
Dear All,
I am thinking that in an SDS-PAGE experiment, if protein samples are
boiled in SDS containing loading dye, and supposedly SDS interacts
with proteins, why the Coomassie Blue dyes could still interact with
and stain the proteins? I am thinking SDS is covering the
proteins, making no room for the Coomassie Blue dyes interaction. I'd
appreciate it if any input from this forum.
Alex
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