A method for global protein expression and antibody screening on high-density filters of an arrayed cDNA library.
We have developed a technique to establish catalogues of protein products of arrayed cDNA clones identified by DNA hybridisation or sequencing. A human fetal brain cDNA library was directionally cloned in a bacterial vector that allows IPTG-inducible expression of His6-tagged fusion proteins. Using robot technology, the library was arrayed in microtitre plates and gridded onto high-density in situ filters. A monoclonal antibody recognising the N-terminal RGSH6sequence of expressed proteins (RGS.His antibody, Qiagen) detected 20% of the library as putative expression clones. Two example genes, GAPDH and HSP90alpha, were identified on high-density filters using DNA probes and antibodies against their proteins.
Max Planck Institute for Molecular Genetics, Ihnestr. 73, D-14195 Berlin, Germany. buessow@mpimg-berlin-dahlem.mpg.de
K
K
Büssow
D
D
Cahill
W
W
Nietfeld
D
D
Bancroft
E
E
Scherzinger
H
H
Lehrach
G
G
Walter
D000911:Antibodies, Monoclonal; D001923:Brain Chemistry; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D005333:Fetus; D015870:Gene Expression; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000276:immunology; D011506:Proteins / Q000235:genetics* / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D009419:Nerve Tissue Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins