separation of phosphorylated and non-phosphorylated peptide

Keld Sorensen KeldS at uic.edu
Fri Jan 3 05:03:44 EST 1997


For kinase assays, most researchers use phosphocellulose
filter paper to trap the peptide. Phosphocellulose is available
in either sheets or as small filter units that fit inside an
Eppendorf tube (Pierce Prod 29520). This works well
for all basic peptides (carrying a positive charge
at low pH) since the interaction is ionic. Note that the
interaction is NOT specific for phosphorylated stuff. 
Phosphocellulose is also available in the format of
a column (Whatman) - you may check with Dima on 
that (do a search on DejaNews for phosphocellulose).

If you are looking for something that specifically 
interacts with phosphorylated stuff, you are looking
at iron-IDA columns or filter papers. IDA matrices
are available from a several manufacturers - 
Pierce sells a complete kit call "Phospho-gel" (Prod 44932)
as well as just the IDA support. Other companies also 
sell just the IDA support. Pierce is also the only 
company that sells iron-IDA paper in the filter buckets 
for the Eppendorf tubes - this system can be used as really
small columns. 

The tradename of the filters in the buckets is
"SpinZyme" - if you do a search on JBC you will
find about 12 or so references using them.

There is also a new product out called INDIA-
phosphoprobe-HRP. It is basically iron-IDA coupled
onto HRP - should work for your westerns etc., but
is not for purification.

It is important to note that all phospho-compounds
will stick to iron-IDA columns, and all basic molecules
will stick to phosphocellulose...... so it is often hard
to separate all the things you want separated in one
session.

Keld Sorensen.



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