Membrane protein purification!!
 

I make the purification of my protein with a Ni-NTA and I quantificate the
protein in UV spectra at 280nm. I have only one peak at 280nm, it's my
protein. After this I take the aliquots with my protein and I use a second
column, High Q. I don't change the buffer and anything. Next, I quantificate
the protein another time in UV spectra at 280nm. In some cases I have two
peaks one at 260nm in the first aliquots and the second at 280nm. In some
aliquots, I have both peaks.
But the peak at 260nm I haven't seen in the first quantification.

I want to know, what is this peak?
I always use a baseline line with the buffer (glyrecol 10%, NaCl100mM, Tris
20mM, B-Mercaptoethanol 5mM, Imidazol 100mM, Melibiose 10mM, DDM 0.1%). This
buffer is the same in the two columns.

Thanks