Novel methods for the detection of protein kinases
Abstract
Several novel techniques have been developed to aid in the characterization, identification, and purification of protein kinases. One of the procedures developed enables the detection of protein kinase activity in protein samples following electrophoresis on SDS-polyacrylamide gels. Proteins are renatured directly in the gel by removal of detergent and, after incubation with ($\gamma$-$\sp{32}$P) ATP, the renatured kinases are allowed to autophosphorylate or phosphorylate substrates polymerized into the gel. The position of radiolabeled proteins can be detected by autoradiography. Another technique involves the ATP affinity analog, 5$\sp\prime$-p-fluorosulfonylbenzoyladenosine (FSBA), which has greatly facilitated the study of protein kinases. An immunochemical approach has been developed for the detection of proteins that have been covalently modified with FSBA, which provides an alternative to the use of radiolabeled ligand. Antibodies have been prepared against FSBA-modified glutamate hydrogenase and purified by chromatography on ATP-agarose. The resulting affinity-purified antibodies react on Western blots with proteins only after they have been FSBA-modified. In addition antibodies have been produced for the detection of proteins labeled with 5$\sp\prime$-p-fluorosulfonylbenzoylguanosine (FSBG), the corresponding GTP affinity analog. Polyclonal antibodies to FSBG-modified glutamate dehydrogenase were affinity purified by chromatography on FSBG-modified polylysine-agarose. On Western blots these affinity purified antibodies reacted with FSBA- or FSBG-modified proteins, but not with unmodified proteins. Characterizations of affinity purified antibodies utilizing ELISAs and Western blots revealed that the anti-FSBA antibodies recognized epitopes localized to the adenosine moiety of the FSBA molecule, whereas the anti-FSBG antibodies interacted with the sulfonylbenzoyl region common to both FSBA and FSBG.
Degree
Ph.D.
Advisors
Geahlen, Purdue University.
Subject Area
Biochemistry
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