Copyright information:Taken from "Two Human ARFGAPs Associated with COP-I-Coated Vesicles"Traffic (Copenhagen, Denmark) 2007;8(11):1644-1655.Published online 29 Aug 2007PMCID:PMC2171037.© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd A) His6-tagged Glo3p lacking its amino terminus was compared with full-length Glo3p as well as full-length Gcs1p in its ability to bind coatomer from yeast cytosol. Protein bound to Ni-NTA agarose beads was resolved by SDS–PAGE, followed by Western blotting and incubation with anti-yeast coatomer antibodies detected by ECL. His-tagged Glo3p lacking its ARFGAP domain (ΔN96) still binds coatomer from yeast cytosol , whereas Gcs1p does not. GTP dissociation inhibitor is used as a negative control here. B) A GST-fusion protein harbouring ΔN96-ARFGAP2 was used for a pull-down from a centrifugation-cleared TX-100 extract of pig brain crude microsomal membranes. Glutathione S-transferase was used as the negative control. Note absence of binding in the negative control and the presence of the characteristic coatomer bands in the pull-down involving the ΔN96-ARFGAP2 GST fusion as the fishing hook. Enrichment of α- and β-COP in the bound fraction was confirmed using antipeptide antibodies (data not shown).