Anti c myc antibody

The Matchmaker™ Gold Yeast Two-hybrid System (Clontech Laboratories, Inc.) was used in accordance with manufacturer's recommendations to analyze the interaction between several reconstructed GAPDH and HIV-1 proteins. Briefly, the bait (cloned into pGBKT7) and prey (cloned into pGADT7) constructs were cotransformed into Y2HGold and plated on QDO/X/A plates (without tryptophan leucine, adenine, and histidine and with aureobasidin A and X-α-Gal). As a positive or negative control, pGADT7-T and pGBKT7-53 or pGADT7 AD and pGBKT7 DNA-BD were cotransformed, respectively. To validate transformed protein expression, each yeast strain was lysed and detected using an anti-HA antibody (Wako Pure Chemical Industries, Ltd.) or an anti-c-Myc antibody (Clontech Laboratories, Inc.).

Cells transfected with pCMV-3Tag-1A-CENPH and pCMV-Myc-GOLPH3 25 (link) were harvested and subjected to co-IP assays. Protein lysates were incubated with 1μg of anti-Flag antibody (Cat#F1804, Sigma-Aldrich, USA) or anti-c-Myc antibody (Cat#631206, Clontech, USA) for 4 h at 4°C under rotation, and then incubated with rProtein A sepharose bead (GE healthcare) for 2 h. The precipitates were washed three times with lysis buffer, and then subjected to Western blot using anti-Flag (1:1000) and anti-c-Myc antibodies (1:500).