Protein complex immunological separation assay (ProCISA): a technique for investigating single protein properties.
Analysis of the posttranslational modification of proteins, such as phosphorylation, might yield misleading results due to the presence of other proteins with similar electrophoretic properties that coimmunoprecipitate with the target protein. The aim of the present work was to develop a reliable, easy and economical technique to completely isolate a protein from its complex. Here we present a new assay developed to fully isolate proteins from macromolecular complexes that consists of an initial SDS/PAGE (under reducing conditions), which isolates the target protein, followed by transfer of the proteins to a buffer, from which the target protein is recaptured by conventional immunoprecipitation. This technique, that we have termed "Protein Complex Immunological Separation Assay" (ProCISA), successfully separated proteins of different sizes, such as pp60Src and the IP3 receptor (IP3R), from their complexes. We show that ProCISA allows the investigation of the tyrosine phosphorylation state of isolated proteins. This technique could also be used to study other posttranslational modifications without risk of misleading results resulting from contamination with other proteins of similar electrophoretic mobility which complex with the protein of interest.
Redondo
P C
PC
Department of Physiology, University of Extremadura, Cáceres 10071, Spain. pcr@unex.es
Rosado
J A
JA
Salido
G M
GM
Sage
S O
SO
D000818:Animals; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000737:chemistry / Q000302:isolation & purification; D046912:Multiprotein Complexes / Q000737:chemistry / Q000302:isolation & purification*; D015688:Oncogene Protein pp60(v-src) / Q000737:chemistry / Q000302:isolation & purification; D015539:Platelet Activation; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000302:isolation & purification*; D013917:Thrombin
D053496:Inositol 1,4,5-Trisphosphate Receptors; D046912:Multiprotein Complexes; D011506:Proteins; D015688:Oncogene Protein pp60(v-src); D013917:Thrombin