A simple improvement in expression cloning.
Expression cloning is an effective approach for isolating genes encoding proteins that associate with a target species. Several molecules have been isolated by expression cloning, including CRE-BP1 associating with Jun (Macgregor et al., 1990); Grb1, identical to p85 PI3-kinase, with the EGF receptor (Skolnik et al., 1991); and Max with Myc (Blackwood and Eisenman, 1991). Expression cloning involves induction of proteins from a lambda gt11 cDNA expression library and screening the proteins on nitrocellulose membranes using a peptide probe (Macgregor et al., 1990). With this method, we previously isolated an Lck tyrosine kinase-associated protein, LckBP1, which is identical to HS1 (Kitamura et al., 1989, 1995; Takemoto et al., 1995). In those experiments, we used a glutathione S-transferase (GST)-Lck SH3 domain fusion protein as a probe, followed by detection of the complex with anti-GST polyclonal antibody. Whereas the ease of obtaining the fusion construct and high-titer anti-GST polyclonal antibody represented clear advantages, the system suffered from high background and low sensitivity. Here we show that pretreatment of nitrocellulose filters with NaDodSO4 reduces background and, in turn, increases sensitivity.
Takemoto
Y
Y
Institute of Immunology, Syntex-Roche, Yamazaki Noda, Chiba, Japan.
Furuta
M
M
Sato
M
M
Hashimoto
Y
Y
D048868:Adaptor Proteins, Signal Transducing; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D051380:GRB2 Adaptor Protein; D011485:Protein Binding; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D020837:SOS1 Protein; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D018076:DNA, Complementary; D005656:Fungal Proteins; D051380:GRB2 Adaptor Protein; D011506:Proteins; D012097:Repressor Proteins; D020837:SOS1 Protein