protein colour problem - (Jul/22/2008 )

Hi All,

I'm wondering if anyone can help.
I have a truncated endolysin i'm working with which purifies perfectly.
However when i'm concentrating the protin after iex using an amicon ultra, the first 14mls to be concentrated turned a yellow colour while the second 14mls to be concentrated did not have a colour change.
I did check the concentration and the one with the yellow colour was 9mg/ml and the other (which did not change) was 7mg/ml.
The protein turned yellow another time during concentration but this was after going through a second round of purification (SEC). (IEX is the first purification procedure I use)
Has this happened to anyone or does anyone have any theories as to what may be happening here??

Thanks !!!

Jen

Sometimes highly-concentrated proteins turn yellow. The reason the second sample wasn't coloured at only a slightly lower concentration could be due to what protein mix you have: if it's a heterogeneous mixture, no single protein will be present at a sufficiently high level to turn the solution, or it could be something about the specific protein in the first fraction.

Do you have a chromatogram of the purification to show us?

[attachment=5051:fplc_SEC_c1.doc]

Hi Swanny,

I attached the chromatogram for the protein which changed colour upon concentration after SEC (the second round of purification) as for IEX we use an econopump not attached to a computer.
About 9mgs of protein was loaded into the FPLC.
We did three runs altogether and pooled the fractions to concentrate them.

Thanks for your help!

Jen