pKLAC2-αAI-OPT

Protocol tips

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	Transformed colonies were transferred onto a replica plate and each colony was grown in 600 µL of YPCGal media in a 96-well microplate at 28 °C for 72–96 h at 250 rpm. Cleared supernatants (200 µL) were loaded into a dot blot apparatus connected to a vacuum pump. Pure Pinto-αAI [2] in sterile YPCGal (1 ng/µL) was used as positive control. Cleared YPCGal inoculated with untransformed cells (200 µL) and sterile YPCGal (200 µL) were used as negative controls.

Protocol tips

Transformed colonies were transferred onto a replica plate and each colony was grown in 600 µL of YPCGal media in a 96-well microplate at 28 °C for 72–96 h at 250 rpm. Cleared supernatants (200 µL) were loaded into a dot blot apparatus connected to a vacuum pump. Pure Pinto-αAI [2] in sterile YPCGal (1 ng/µL) was used as positive control. Cleared YPCGal inoculated with untransformed cells (200 µL) and sterile YPCGal (200 µL) were used as negative controls.

Publication protocol

Transformed colonies were transferred onto a replica plate and each colony was grown in 600 µL of YPCGal media in a 96-well microplate at 28 °C for 72–96 h at 250 rpm. Cleared supernatants (200 µL) were loaded into a dot blot apparatus connected to a vacuum pump. Pure Pinto-αAI [2] in sterile YPCGal (1 ng/µL) was used as positive control. Cleared YPCGal inoculated with untransformed cells (200 µL) and sterile YPCGal (200 µL) were used as negative controls. The nitrocellulose membrane was probed with a rabbit anti-αAI polyclonal antibody (1:10,000 dilution), and then probed with HRP-conjugated anti-rabbit secondary antibody (1:1000 dilution, Promega Corp. Madison, WI, USA). Protein antibody complexes were made visible using Pierce ECL Western Blotting Substrate detection reagent (Thermo-Fisher Scientific, Inc., Waltham, MA, USA) and autoradiography film (Kodak Biomax X-ray film). The relative amount of protein in dots was estimated by densitometry using ImageJ Software [38].

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