Self-assembling protein microarrays.
Protein microarrays provide a powerful tool for the study of protein function. However, they are not widely used, in part because of the challenges in producing proteins to spot on the arrays. We generated protein microarrays by printing complementary DNAs onto glass slides and then translating target proteins with mammalian reticulocyte lysate. Epitope tags fused to the proteins allowed them to be immobilized in situ. This obviated the need to purify proteins, avoided protein stability problems during storage, and captured sufficient protein for functional studies. We used the technology to map pairwise interactions among 29 human DNA replication initiation proteins, recapitulate the regulation of Cdt1 binding to select replication proteins, and map its geminin-binding domain.
Ramachandran
Niroshan
N
Harvard Institute of Proteomics, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 320 Charles Street, Cambridge, MA 02141, USA.
Hainsworth
Eugenie
E
Bhullar
Bhupinder
B
Eisenstein
Samuel
S
Rosen
Benjamin
B
Lau
Albert Y
AY
Walter
Johannes C
JC
LaBaer
Joshua
J
D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002474:Cell-Free System; D004261:DNA Replication; D018076:DNA, Complementary; D000939:Epitopes; D064248:Geminin; D006801:Humans; D064111:Minichromosome Maintenance Complex Component 2; D064151:Minichromosome Maintenance Complex Component 6; D009687:Nuclear Proteins / Q000378:metabolism; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods; D011485:Protein Binding; D014176:Protein Biosynthesis; D025941:Protein Interaction Mapping / Q000295:instrumentation / Q000379:methods; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D018741:Replication Origin; D014158:Transcription, Genetic
C467199:CDT1 protein, human; D018797:Cell Cycle Proteins; D018076:DNA, Complementary; D000939:Epitopes; C577995:GMNN protein, human; D064248:Geminin; D009687:Nuclear Proteins; D011506:Proteins; C084859:MCM2 protein, human; C103258:MCM6 protein, human; D064111:Minichromosome Maintenance Complex Component 2; D064151:Minichromosome Maintenance Complex Component 6