In article <22.214.171.12400128092822.00a2b850 at facstaff.wisc.edu>, Debora
Fontanini <dfontani at facstaff.wisc.edu> writes
>The column volume is about 15 ml and I do not think that the gradient is steep
>at all for this volume. I am going to 40% B in 30 minutes and my sample comes
>off in the middle of the gradient.
Fine but what volume is the gradient? 10x column volume is pretty
standard i.e. 150ml. Washes at 100% B should be at least 3 column
volumes etc. to equilibrate the column in high salt then 3 column
volumes at low salt prior to loading again.
I can't remember but you should be able to put NaOH through that column
which should strip it and clean it pretty well. The only thing that
really kills or at least messes with binding to mono Q (again from
memory) is metal ions which is why the original FPLC had no metal
pipework unlike standard HPLC's of the time.
Can you resolve a crude E.coli extract or a set of standard proteins as
per FPLC literature or whatever on your monoQ. Basically ignore what
your protein is doing and find something simple that will show that the
monoQ is behaving correctly or not. Having proved that it is OK one can
then look more closely at your sample.
Another thing to try is to get hold of some prepacked FFQ sepharose in
one of say the inexpensive 5ml columns that APB sell. The resolution
will not be as good as the monoQ but the elution characteristics of the
column should be pretty much identical so your protein should elute in
the middle of your gradient at the same salt molarity as per your
original monoQ. Again running a standard set of proteins on this and the
monoQ should calibrate both columns and then running your protein will
give more idea as to what is going on.
I'm afraid it looks like more work is required.
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....