Dear netters,
we have done overexpression of a cytosolic
hemoprotein in E. coli, got nice expression and
have done DEAE anion exchange chromatography
of the ultracentrifuge supernatant as first
purification step. During the elution with
a buffer containing 50mM Hepes pH 7.4, NaCl
450 mM, DTT 5mM, Pefabloc 1 mM (Merck, PMSF
like stuff) we have noticed brownish stuff at
the tip of the elution tube. We have taken
some stuff from the tip onto a glass slid
and under the microscope
there were apparent big NaCl crystals
and much smaller yellow - brownish crystals.
The fractions containin our protein looked nice
but there were still some contaminating proteins.
I would say maybe 70 % purity. Protein conc. was
2.5 mg / ml.
I know that you normally need much higher protein
purity and concentration to get protein crystals.
Does DTT form brown crystals or could it be nucleic
acids or something else non-protein like from the
coli?
On the other hand could the brown yellowish color
be compatible with a hemoprotein? And maybe the
protein under investigation just crystallizes like
hell?
I would be very happy about any advice or comment
or guess or hint on literature.
Thanks a lot for your time and help
Soenke
