Criterion tgx polyacrylamide gel

Cells were lysed in lysis buffer containing 1% NP-40, 50 mM HEPES (pH 8.0), 5 mM EDTA, 10 mM sodium pyrophosphate, 50 mM NaF, 0.25 % sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM benzamidine, 1 µg/ml pepstatin, 10 µg/ml leupeptin, 20mM iodoacetimide, and 100 µg/ml soy bean trypsin inhibitor. The lysates were centrifuged at 14,000 rpm for 15 min at 4 °C. RIPA buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% Triton-X-100, 1% NP40, and 0.1% SDS) was used for the detection of sex hormone receptors. The protein concentration of the supernatants were then measured using the Pierce BCA protein assay reagents and 25 µg of protein was loaded in each well of either 10% or 4–20% Criterion TGX polyacrylamide gels (Biorad, Hercules, CA). The gels were transferred onto a 0.2 mm PVDF membrane that was then probed with antibodies against YM1, ARG1, FIZZ1, and β-actin. Secondary antibodies conjugated to HRP were used to detect target proteins, which was visualized with ECL reagent and autoradiograph film. Lightly exposed x-ray films were used for densitometric analysis. Films were scanned and band intensities were measured using ImageJ software (NIH). Target proteins were normalized to β-actin internal control. To assess fold changes in signal, the target protein/β-actin values were then normalized to the control group within each experiment.

Lipid II used to monitor glycosyltransferase activity of the protein complex was dried using a nitrogen stream and dissolved in an equal volume of dimethyl sulfoxide (DMSO). The reaction and detection of glycan strands was adopted from previously published protocols^{3 (link),23 (link),42 (link),56 (link)}. Briefly, PaFtsWIQLB and EcFtsWIQLB were mixed at a final protein concentration of 1 µM in 10µL with 1 µL 10x Reaction Buffer (500 mM Tris pH 7.5, 200 mM MnCl₂), 1 µL DMSO and 1 µL Lipid II and incubated for 30 min at 25°C. Proteins were heat inactivated for 2 min at 95°C. Lipid II and glycan strands were labelled by incubating the reaction with 26 µM SaPbp4 and 20 mM BDL for 1 h at 25°C. An equal volume of Laemmli SDS-PAGE buffer was added and the mixtures were heat inactivated for 3 min at 95°C. Glycan strands were separated from Lipid II on a 4-20% Criterion TGX polyacrylamide gel (Bio Rad), run for 45 min at 200 V. After blotting onto PVDF membrane, the blot was incubated for 2 h in Superblock blocking buffer TBS (Thermo Scientific), followed by incubation with a 1:5000 dilution of IRDye800CW Streptavidin (LI_COR Bioscience) in TBS buffer at room temperature for 1 h. The blot was washed three times in PBS buffer and bands were visualised using an Odyssey CLx imaging system (Li-COR Bioscience).