Blot overlays with 32P-labeled fusion proteins.
Proteins labeled with 32P can be used as sensitive "prime" in blot overlays to detect binding proteins or domains. Small G-protein Ras can bind GTP with extremely high affinity (Kd approximately 10(-11)-10(-12) M) in the presence of Mg2+. We have taken advantage of this property of Ras to develop a vector that expresses proteins of interest such as glutathione S-transferase (GST)/Ras fusion proteins for noncovalent labeling with [gamma-32P]GTP. The labeling efficiency of this method is >60% and involves a single short incubation step. We have previously identified several binding proteins for the second SH3 domain of the adaptor Nck using this method. Here we illustrate the overlay method using the GST/Ras system and compare results with the SH3 domain labeled by phosphorylation with [gamma-32P]ATP. Both methods are similarly specific and sensitive; however, we show that signals are dependent primarily on GST-mediated probe dimerization. These dimeric probes allow a more stable probe-target complex similar to immunoglobulin interactions, thus significantly improving the sensitivity of the technique.
Zhao
Z
Z
Glaxo-IMCB Group, Institute of Molecular & Cell Biology, 30 Medical Drive, 117609, Singapore.
Lim
L
L
Manser
E
E
D048868:Adaptor Proteins, Signal Transducing; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000502:physiology*; D006160:Guanosine Triphosphate / Q000378:metabolism; D015335:Molecular Probes; D015513:Oncogene Proteins / Q000502:physiology; D015514:Oncogene Proteins, Fusion / Q000378:metabolism; D010761:Phosphorus Radioisotopes / Q000378:metabolism; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D015800:Protozoan Proteins / Q000378:metabolism; D011869:Radioligand Assay; D051381:Rats; D018909:src Homology Domains / Q000502:physiology
D048868:Adaptor Proteins, Signal Transducing; D015335:Molecular Probes; C078332:Nck protein; D015513:Oncogene Proteins; D015514:Oncogene Proteins, Fusion; D010761:Phosphorus Radioisotopes; D015800:Protozoan Proteins; C424065:rasG protein, Dictyostelium; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D005982:Glutathione Transferase