Purification and functional properties of prenylated Rab acceptor 2.
PRA2 was found to interact with the ER-localized protein VAMP-associated protein of 33 kDa or VAP-33 by a yeast two-hybrid screen. We describe here the purification of PRA2 and VAP-33 as well as an in vitro pull-down procedure to verify the interaction. PRA2 was found to form a large sodium dodecyl sulfate (SDS)-insoluble complex upon heat denaturation, resulting in significant reduction in the Western immunoblot signal. This phenomenon is specific to PRA2 and was not observed with PRA1. We also found that protein interaction with PRA2 is highly sensitive to detergent and describe a covalent cross-linking procedure for mammalian cell extracts to stabilize the PRA2-containing complex prior to membrane solubilization and immunoprecipitation.
Gougeon
Pierre-Yves
PY
Ngsee
Johnny K
JK
D001483:Base Sequence; D017931:DNA Primers; D019204:GTP-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology*; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology*; D010957:Plasmids; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology*
D017931:DNA Primers; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D033921:Vesicular Transport Proteins; D005982:Glutathione Transferase; D019204:GTP-Binding Proteins; C108941:RABAC1 protein, human