Monoclonal anti gfp

Allelic replacement of the genes encoding the alpha (Msmeg_3178) or beta (dnaN, Msmeg_0001) subunits of DNA polymerase III with the alpha-eyfp or dnaN-mCherry fusion genes, as well as replacement of the parB gene with the parB-mNeon green or parB-mCherry fusion genes, was performed as previously described (47 (link), 48 (link)). Replacement of the hupB gene with hupB-egfp was performed as described by Hołówka and coworkers (49 (link), 50 (link)). The mycobacterial strains were cultured in 37°C in 7H9 broth or on 7H10 agar (Difco) supplemented with oleic acid-albumin-dextrose-catalase (OADC; BD), 0.05% Tween 80, and (when needed) proper additives (kanamycin at 50 μg/ml, X-Gal [5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside], 2% sucrose). Correct allelic replacement and proper incorporation of the integration vectors were confirmed using PCR and Western blotting. Fusion of the functional fluorescent protein was confirmed by seminative SDS-PAGE and was visualized using a Typhoon phosphorimager. Western blotting was performed using standard procedures (77 ) with polyclonal anti-mCherry, monoclonal anti-GFP (Santa Cruz Biotechnology), and monoclonal anti-mNeon (Chromotec) antibodies.

Twenty-four hours post induction, cellular extracts of stable transgenic HeLa expressing SPLICS_S/L-P2A probes were western blotted to evaluate GFP_1–10 or YFP_1–10 protein expression. Cells were lysed in RIPA Buffer (50 mM Tris-HCl pH 7.4, NaCl 150 mM, 1% Triton X-100, 0.5% sodium deoxycholate, 10 mM EDTA, 0.1% SDS, 1 mM DTT, 2× Protease Inhibitor Cocktail (Merck KGaA, Darmstadt, Germany, Cat# P8340)) for 20 min. Extracted proteins were quantified by Bradford assay (Bio-Rad, Hercules, California, USA Cat# 500-0205), resolved on SDS-PAGE in 12% SDS/PAGE Tris- HCl polyacrylamide gel, and then transferred to PVDF membranes (Bio-Rad, Hercules, California, USA) using Trans-Blot^® Turbo™ Transfer System (Bio-Rad, Hercules, California, USA). Membranes were blocked with 5% w/v non-fat dried milk (NFDM) in TBST (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween-20) and incubated overnight with the specific primary antibody at 4 °C. Signal was detected by incubation with secondary horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibodies for 1 h at room temperature followed by incubation with the chemiluminescent reagent Luminata Classico HRP substrate (Merck Millipore, Cat# WBLUO500), monoclonal anti-GFP 1:500 (Santa Cruz, Cat. 9996) and monoclonal anti-ßeta Tubulin 1:2000 (Cell Signalling, Cat. 2128).

Proteins were separated by 15% SDS-PAGE and transferred to 0.2 μm PVDF membranes (Bio-Rad, CAD) for 1 h at 0.4 V cm⁻¹ in a Mini-PROTEAN® Tetra Cell (Bio-Rad, CAD). His-tagged proteins were detected using 1:1000 diluted monoclonal anti-polyHis-HRP conjugate (Sigma-Aldrich, CAD). GFP and GST tagged proteins were visualized with 1:1500 diluted monoclonal anti-GFP and anti-GST-HRP conjugates, respectively (Santa Cruz Biotechnology, USA). Biotinylated proteins were detected with 1:2000 diluted HRP-conjugated Streptavidin (Thermo Fisher Scientific, CAD). Membranes were blocked with 5% skim-milk (BD Difco, CAD) and HPR activity was detected using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, CAD) with a ChemiDoc™ Imaging System (Bio-Rad, CAD) able to detect the chemiluminiscent signal.