Cytohesin pull-down assay

To quantify GEP activity of cytohesins in vitro, most assays measure ARF binding of guanine nucleotides using either radiolabeled nucleotides (e.g., Pacheco-Rodriguez et al., 1998) or changes of the tryptophan fluorescence of ARF that occur when bound GDP is replaced by GTP (e.g., Beraud-Dufour et al., 1998). GEP action on ARFs in cells has been evaluated by pull-down assays to recover activated ARF bound to GGA proteins (Santy and Casanova, 2001). 

Detection of Cytohesin 2 and IPCEFl Interaction Using GST-fusion Protein Pull-Down Assay... 

The principle of pull-down assay is based on the ability and specificity of a protein of interest, immobilized via its tag to an affinity matrix, to precipitate or pull-down its partner from the cell lysates. The glutathione S-transferase (GST) fusion protein pull-down approach is a commonly used method, in which the GST serves as a tag when fused to the protein of interest, and glutathione beads serve as the pull-down matrix. This section describes the use of GST pull-down assay to evaluate the interaction between cytohesin 2 and IPCEFl. This assay involves expression and purification of the GST-fusion protein in bacteria and expression of the interactor protein with an epitope-tag in mammalian cells, incubation of GST-fusion protein coupled to glutothione matrix with a lysate of cells expressing the epitope-tagged interactor protein, and analysis the protein complex by Western blotting. 

IPCEFl has been reported to interact with ADP-ribosylation factor GTP exchange factors of the cytohesin family and function by modulating the cytohesin 2 activity. This article describes methods used to study the interaction and activation of cytohesin GEFs by IPCEFl. The experimental approaches described here include physical and functional interaction assays by which the association of IPCEFl with cytohesin 2 is explored both in vitro and in vivo. The methods used to analyze the physical association include GST-pull down and coimmunoprecipitation approaches. We also used yeast two-hybrid and colocalization assays to study the interaction between IPCEFl and cytohesins. The functional relationship between IPCEFl and cytohesin 2 was assessed by studying the effect of IPCEFl on the in vitro and in vivo stimulation of ADP-ribosylation factor 6 GTP formation by cytohesin 2. 

---