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Dear Sergei,
Of course I did not want to say that coiled-coils cannot be a subject
for GF. I just have an experience with a coiled-coil containing protein
going in the dead volume without any aggregation.
Best regards,
Julia
Sergei Strelkov wrote:
Dear Julia,
Does your protein contain a coiled-coil? If yes, it does not mean
that it is aggregated - it simply cannot enter the pores in the GF
media.
I would be cautious here. We are working with a number
of coiled-coil fragments which are most typically
about 80 residues long (some are longer). A coiled-coil
of 80 residues is a 12nm long rod. They are expressed
with an N-terminal 6xHis tag which is cleaved off after the
purification of the Ni column. As the final step, we use gel-filtration
on Superdex 75. Most fragments apparently do enter the column
and yield a decent peak (these are double-stranded coiled coils).
However, we did see aggregation with some of our fragments.
In this case there is indeed a peak or a family of peaks
close to the void volume.
Best wishes,
Sergei
