dimers and PTPase activity
hlorenzo at pasteur.fr
Thu Nov 21 11:42:52 EST 1996
I have a problem measuring the activity of a protein-tyrosine-phosphatase.
I use a recombinant protein which includes MBP (Maltose-binding-protein) as fusion protein (~40 kDa) and a PTPase (~36 kDa). Affinity chromatography in amylose resin + MonoQ yields a 99% of pure protein.
My problem after purification consists that the initial PTPase activity (using pNitrophenylphosphate as substrate) quickly drops, probably due to dimer/oligo/merization (I've recognized multimeric forms by gel filtration but always monomeric after SDS PAGE).
My interest, however, resides in measuring the PTPase activity from the monomer of the entire protein. I've achieved to recover and stabilize the monomer in presence of different detergents (ranging 0.05-0.1% ) contained in classic PTPase buffers (f.e. 50 mM Imidazole pH 7.0 + 150 mM NaCl + 0.1% BME). Unfortunately, the resulting changes in the protein conformation brings about a PTPase activity almost or even undetectable.
Help or comments from anyone with similar problems will be greatly appreciated.
More information about the Proteins