Golgins assay

TETHERING ASSAYS FOR COPI VESICLES MEDIATED BY GOLGINS... 

In Vitro COPI Vesicle Tethering Assay Using Purified Golgins... 

The in vitro transport assay is routinely characterized to ensure that the assay has indeed faithfully reconstituted the EE/RE-to-TGN transport in vitro (Tai et ah, 2004). First, the assay must be cytosol-dependent. The basal level of transport in the absence of cytosol is usually 5-10% of that in the presence of 1.2 mg/ml of rat liver cytosol. Second, the assay is tested with some commonly used inhibitors such as Brefeldin A and guanosine 5 -0-(3-thio) triphosphate (GTP7S), as their inhibitory role in EE/RE-to-TGN traffic is well documented. Finally, the assay is tested using antibodies (such as autibody agaiust syutaxiulb) or recombinant proteins (such as GST-GRIP of golgin-97) that have been shown to inhibit in vitro EE/RE-to-TGN transport of STxB. 

Fig. 1. The recombinant GRIP domain of golgin-97 inhibits in vitro EE/RE-TGN transport. STxB transport assays are carried out in the absence (lanes 1 and 4) or presence of 133 g/ml of GST-GRIP domain proteins (lane 2, GST-GRIP lane 3, heat-denatured GST-GRIP lane 5, GST-GRIPA 697A). Extent of transport relative to controls (set as 100%) is calculated from two to three independent experiments. Error bars represent standard deviations. Bottom panel shows representative phosphor-imaging data. Reprinted from Molecular Biology of the Cell (Mol. Biol. Cell (2004). 15, 4426-4443.) with the permission of The American Society for Cell Biology.

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