Abstract

In the current post-genomic era, large scale efforts are underway to functionally explore the proteome by assembling large antibody libraries. However, because many proteins are modified post-translationally to regulate their function, collections of modification-specific sensors are also needed. Here we applied a novel approach to select monoclonal phosphospecific antibodies directly from the full-length protein and without up-front phosphoamino acid identification. We chose as antigen GRASP65, a well studied Golgi phosphoprotein. Bacterially produced full-length protein was first incubated with mitotic cytosol, thus allowing modification by naturally occurring kinases, and then used directly for affinity-based antibody selection using a single chain variable fragment phagemid library. In less than 1 week, three distinct and highly functional monoclonal phosphospecific antibodies against two GRASP65 epitopes were obtained and subsequently characterized. The presented approach is carried out fully in vitro, requires no prior knowledge of the phosphoamino acid identity, and is fast and inexpensive. It therefore has great potential to be an attractive alternative to classic animal-based protocols for the selection of post-translation modification sensors and thus to become an invaluable tool in our quest to understand the proteome in all its complexity.

Highlights

  • Cancer” network (2) as well as commercial enterprises are currently sponsoring large projects to assemble all-inclusive antibody libraries

  • This study describes a novel approach, which allowed the direct selection and characterization of three distinct and highly functional monoclonal phosphospecific antibodies

  • Full-length Antigen Is Synthesized in Bacteria and Directly Phosphorylated—Classical methods to obtain phosphospecific antibodies require the design and subsequent synthesis of highly specific short phosphopeptides, which are used for animal immunization

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Summary

EXPERIMENTAL PROCEDURES

Antigen Preparation—Poly-His-tagged full-length rat GRASP65 was expressed in Escherichia coli and purified on Ni-NTA6 beads as described (12).

Direct Selection of Phosphospecific Antibodies
RESULTS
DISCUSSION
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