Anti pgk1 antibody

Protein samples were prepared using an alkaline method and resolved by 10% SDS–PAGE. Anti-Myc antibody was purchased from Covance (Madison, WI); anti-Flag antibody was from Sigma-Aldrich (St. Louis, MO); anti-GFP antibody was from Santa Cruz Biotechnology (Santa Cruz, CA); and anti-Pgk1 antibody was from Molecular Probes (Eugene, OR). The horseradish peroxidase–conjugated goat anti-mouse immunoglobulin G secondary antibody was purchased from Jackson ImmunoResearch (West Grove, PA).

At each timepoint, three OD eq of cells were harvested and centrifuged at 14,000 × g for 2 min. Cell pellets were then resuspended in 100 µl SUME buffer (1% SDS, 8 M urea, 10 mM MOPS, 10 mM EDTA, pH 6.8) with protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 260 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 100 mM leupeptin hemisulfate, 76 mM pepstatin A, 5 mM 6-aminocaproic acid, 5 mM benzamidine, and 142 mM TPCK). Silica beads were then added, and cells were lysed on a multivortexer (1 min of vortexing at room temperature followed by 1 min on ice, repeated three times). A 100 µl of 2X urea sample buffer (8 M urea, 4% SDS, 200 mM dithiothreitol, 125 mM Tris, pH 6.8) was added to each lysed sample, and the mixture was then boiled at 95°C for 8 min. Finally, samples were centrifuged at 14,000 × g for 5 min.
In all cases, samples were resolved on 10% acrylamide gels by SDS–PAGE, transferred to nitrocellulose in 13% methanol, and blotted with mouse monoclonal anti-GFP antibody (Living Colors) or anti-PGK1 antibody (Molecular Probes) followed by goat anti-mouse HRP-conjugated secondary antibody (Jackson ImmunoResearch).

Three OD eq cells were harvested by centrifugation at 14,000 × g for 5 min. Pellets were resuspended in 100 µl SUME (SDS, urea, MOPS, ETDA) buffer (1% SDS, 8 M urea, 10 mM MOPS, 10 mM EDTA, pH 6.8) with protease inhibitors (PIs) (1 mM phenylmethylsulfonyl fluoride, 260 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 100 mM leupeptin hemisulfate, 76 mM pepstatin A, 5 mM 6-aminocaproic acid, 5 mM benzamidine, and 142 mM tosyl phenylalanyl chloromethyl ketone [TPCK]), and 0.5 mm glass beads were added to the meniscus. Cells were lysed three times at 1-min intervals on a multivortexer at room temperature with 1 min on ice in between. After the addition of 100 µl 2× urea sample buffer (2× USB: 8 M urea, 4% SDS, 200 mM dithiothreitol [DTT], 125 mM Tris, pH 6.8), samples were heated at 95°C for 10 min and clarified by centrifugation at 14,000 × g for 5 min. Samples were resolved by SDS–PAGE, transferred to nitrocellulose in 12% methanol, and blotted with mouse monoclonal anti-GFP antibody (Living Colors), anti-HA antibody (Thermo Fisher Scientific), or anti-PGK1 antibody (Molecular Probes) (loading control) followed by goat anti-mouse HRP-conjugated secondary antibody (Jackson ImmunoResearch).