Detection of phosphorylation in non-denaturing condition

Phil Robinson mdpjr at cc.newcastle.edu.au
Mon Jul 8 20:34:50 EST 1996


dykim at kaist.ac.kr (Dooyeon Kim) wrote:
>Is there any ways to seperate the phosphorlated protein from non-phosphoryl
>ated form in non-denaturing condition? I am thinking of using non-denaturing
>electrophoresis of iso-electric focusing in non-denaturing condition but I
>have no reference about these things. Any information or suggestions will be 
>greatly appreciated. Please help me.

You should not need much help to try IEF, there are plenty of methods.
An overlooked method is metal chelate chromatography, which works for
lots of phosphoproteins.  Could be of help.

Immobilised Metal Ion Affinity Chromatography (IMAC) can be used
especially for enrichment of phosphorylated proteins, peptides and
amino acids.  The method is based on the binding of metal ions to a
resin of iminodiacetic acid (IDA), which is coupled by ether links to
agarose.  Commercially available as IDA-agarose from Sigma (I4758),
Pierce (PhosphoGel kit, 44932G) or IDA-sepharose (Pharmacia, or
Sigma).  The resin is charged with Fe3+ (other metals are used for
other applications).  Then the proteins are affinity bound to the
activated matrix, forming a chelation complex under phosphate-free,
acidic conditions.  The bound proteins may be eluted by addition of
phosphate ions, Mg2+, phosphoserine, and/or increasing the pH.
Proteins, amino acids, and peptides that contain phosphoserine,
phosphotyrosine and/or other phosphothreonine residues bind very
strongly to the Fe3+-activated matrix.  One of the strongest known
bonds occurs between phosphoserine and ferric ions; the binding
constant is apparently greater than 10-13.  The strength of binding is
related to the number of phosphorylated amino acids, resulting in
higher pH required for elution of multiply phosphorylated proteins.
1.    Anderson, L.,and Porath J.  (1986) Analytical Biochemistry 154,
250-254.
2.    Porath, J., et al (1983) Biochemistry 22, 1621.
3.    Toomik, R., et al (1993).  Alanlytical Biochemistry, 209,
348-353.
4.    Erickson, A.C. and Johnson, G.V.W. (1993). J. Neurosci. Meth.
46, 245-249.

Phil				Newcastle,  Australia




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