Mouse anti gfp first antibody

Ribosome quality control reporter was tested as described (31 (link)). The WT, C719A, and G906D strains and their ltn1Δ, rqc1Δ and rqc2Δ strains carrying the pTDH3-GFP-R12-RFP were grown in SD–Ura. Cell pellets were resuspended in 200 μl lysis buffer (PBS pH7.0, 10 mM DTT, 1 mM PMSF, 100× protease inhibitor cocktail, and 3 mg/ml zymolyase), and incubated at room temperature for 30 min with 15 min intervals for vortexing. After sonication and centrifugation (16 000 g for 5 min), supernatant proteins were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes. Next, standard western blotting procedure was followed to detect the GFP levels. The mouse anti-GFP first antibody (Invitrogen) with 1:1000 dilution and goat anti- mouse IgG-HRP secondary antibody (Invitrogen) with 1:5000 dilution were used. Nitrocellulose membranes were treated with ECL chemiluminescent substrate reagents (Bio-Rad) and visualized using a ChemiDoc Imaging System (Bio-Rad).

Yeast cells carrying pTDH3-GFP-R12-RFP(34) were grown in SD-Ura. Cell pellets were resuspended in PBS and adjusted to the same A₆₀₀. The total proteins were extracted as described above and separated on a 12% SDS-PAGE gel. A standard western blot protocol was applied as described above. The mouse anti-GFP first antibody (Invitrogen) was used with 1:1000 dilution.