Dear Goran: While HPT works (principally by magic) or a derivative
thereof, elution can be acheived by changes in ionic strength as well as
(sometimes) by an affinity ligand. For example, some phosphatases will
stick very tightly to HPT and can be eluted in non-phosphate buffers. I
have also seen reports of using a NaCl gradient on HPT, although I cannot
vouch personally for success. Interaction is mainly through, ionic, some
matrix-type and also through a quasi-affinity for phosphates. e.g.,
Several reductases will also stick very tightly to HPT at low phosphate
concentrations. You could avoid phosphate and use a Goods buffer
containing calcium chloride. Glycoprotein interactions to HPT might be
modulated by changing the initial ionic strength. In my hands, principall=
y
with plant extracts, the behaviour of many proteins on HPT is generally
reflective of their behaviour on phenyl-speharose. For instance,
hydrophilic proteins do not bind to HPT at a phosphate concentration abov=
e
50 to 60 mM, they are also readily eluted from phenyl-sepharose in 0.6 to
0.9 M ammonium sulfate, if they are loaded on a phenyl-sepharose column
equilibarted with 1.8M ammonium sulfate. This behaviour of hydrophilic
proteins (I think also glycoproteins and those with a high degree of
alpha-helices) should be eluted from HPT using salt or potentially even a
sugar or a cheap sugar-phosphate. I will stop here before I dig too deep=
a
hole. Hope this helps, good luck, gautam
Gautam Sarath
N-226, Beadle Center
Protein Core Facility - Center for Biotechnology &
Department of Biochemistry
University of Nebraska-Lincoln
Lincoln, NE 68588-0664
Phone: 402-472-2928
FAX: 402-472-7842
http://www.biotech.unl.edu/Proteins/index.html