The following commercially available antibodies were used for Western blotting: mouse monoclonal antibodies against clathrin heavy chain (CHC; BD Biosciences; 610500), lamin A/C (Santa Cruz Biotechnology; sc-7292), Hsp90 (Santa Cruz Biotechnology; sc-13119), EHD2 (Santa Cruz Biotechnology; sc-100724), dynamin (BD Biosciences; 610245), and filamin A (Chemicon; MAB1678); rabbit polyclonal antibodies against SUMO2/3 (Cell Signaling Technology; 4971), Cav1 (BD Biosciences; 610059), pacsin2 (Abgent; AP8088b); and cavin1 (Sigma; AV36965); for immunofluorescence, mouse monoclonal anti-cavin1 (BD Biosciences; 611258), goat polyclonal anti-EHD2 (Abcam; Ab23935), rabbit polyclonal anti-Cav1 (BD Biosciences; 610059), mouse monoclonal anti-lamin A/C (Santa Cruz Biotechnology; sc-7292), rabbit polyclonal anti-SUMO1 (Cell Signaling Technology; 4930), and rabbit polyclonal anti-SUMO2/3 (Cell Signaling Technology; 4971). Antibodies conjugated to Alexa Fluor 488, Cy3, Cy5, or HRP (Beckman Coulter and Invitrogen) were used as secondary antibodies. HaloTag dye JF635 was provided by L. Lavis (Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA). Accutase was purchased from Sigma-Aldrich. Hepes, SDS, and Tris were purchased from Euromedex.
Sodium dodecyl sulfate (sds)
Manufactured by Euromedex
The SDS is a laboratory equipment used for the separation and analysis of proteins and other macromolecules. It operates on the principle of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which is a technique used to separate proteins based on their molecular weight.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Roche (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Dithiothreitol (dtt), by Merck Group (2 mentions)
Anti sumo1, by Cell Signaling Technology (1 mentions)
Anti sumo2 3, by Cell Signaling Technology (1 mentions)
Hepes, by Euromedex (1 mentions)
Anti cav1, by BD (1 mentions)
Accutase, by Merck Group (1 mentions)
Sds page gel, by Bio-Rad (1 mentions)
Chemidoc touch biorad imager, by Bio-Rad (1 mentions)
β mercaptoethanol, by GE Healthcare (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Image lab, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Roche (1 mentions)
Tris hcl ph 8, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Euromedex (1 mentions)
Edta ph 8, by Thermo Fisher Scientific (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
Bioruptor pico, by Diagenode (1 mentions)
5 protocols using sodium dodecyl sulfate (sds)
1
Western Blotting and Immunofluorescence Antibodies
2
Western Blot Sample Preparation and Analysis
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1 million cells were lysed in 100μl of LBX in presence of cOmplete EDTA free Protease inhibitor cocktail (Roche). The lysates were sonicated for 20 minutes at 4°C in a Sonorex Digitec (model DT100) ultrasonic bath (Bandelin) and cleared by centrifugation at 16,000 g for 10 minutes at 4°C. The supernatant was stored at −20°C until blotting. 6X Laemmli buffer (12% SDS (v/v) (Euromedex), 58% Glycerol (v/v) (Pharmacia Biotech), 375mM Tris HCl pH 6.8, 30% β-mercaptoethanol (v/v) (Pharmacia Biotech), 0.0012% Bromophenol Blue Before (w/v) (Pharmacia Biotech)) was added to samples to a final concentration of 1X prior to gel run. Samples were boiled at 95°C for 20 minutes on a thermoblock, immediately chilled on ice and centrifuged at 16,000 g for 5 minutes. 15-40μl of samples were resolved on 4%–20% SDS-PAGE gels (Biorad) and transferred on nitrocellulose membrane (Biorad). Membranes were saturated and proteins were blotted with antibodies (listed in Key Resources Table ) in 5% non-fat dry milk, PBS, 0.1% Tween buffer. ECL signal was recorded on a ChemiDoc Touch Biorad Imager. Data was analyzed with Image Lab (Biorad).
3
Cross-linking and Chromatin Preparation
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10 million cells were cross-linked in medium with 1% formaldehyde for 8 min at RT on a slow shaker, quenched with freshly prepared 0.125M glycine, incubated 5min at RT on a slow shaker, then pelleted at 400 g for 5 minutes at 4°C, washed three times with 30ml of ice cold PBS and then incubated for 20 minutes rotating at 4°C in 1mL of RIPA lysis buffer (10mM Tris-HCl pH 8.0, 1mM EDTA pH 8.0 (Invitrogen), 140mM NaCl, 1% (v/v) Triton X-100 (Euromedex), 0.1% (v/v) SDS and 0.1% sodium deoxycholate (SIGMA)). Nuclei were pelleted at 1350 g for 5 minutes at 4°C, washed for 10 minutes rotating with 1ml of a buffer containing 10mM Tris, 200mM NaCl, 1mM EDTA (Invitrogen), 0.5 mM EGTA (Euromedex), pelleted and lysed in buffer containing 0.4% SDS (Euromedex), 10mM EDTA (Invitrogen), 50mM Tris-HCl pH 8.0 for 30min on ice (volume of buffer = 100μl/1.6 million cells). Lysates were sonicated on a Bioruptor Pico (Diagenode) sonication devices (11cycles 30 s ON, 30 s OFF) to reach fragments ranging from 150 to 500bp, and then centrifuged at 10,000 g for 10 minutes at 4°C to remove debris. Samples were then snap-frozen in liquid nitrogen and stored at −80°C until immunoprecipitation. All buffers contained cOmplete EDTA free Protease inhibitor cocktail (Roche).
4
Western Blotting Reagent Protocol
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Tris, glycine, SDS, dimethylsulfoxide (DMSO), acrylamide and bis-acrylamide were purchased from Euromedex. Ponceau S, p-coumaric acid, bromophenol blue, brilliant blue R250, MgSO4, Ca(NO3)2, COSO4, CuCl2, ZnSO4, Na2M0O4, Na2B4O7, FeSO4, VOSO4, vitamins, HEPES, hydrogen peroxide, Tween 20, luminol, Triton X-100, sucrose, acetic acid, dithiothreitol (DTT), RNase A, and propidium iodide (PI) were obtained from Sigma. KCl, H2SO4 and KH2PO4 were from Merck. NaCl was from Fischer scientific. Tryptone peptone and yeast extract were obtained from Difco (Becton Dickinson). Ammonium persulfate (APS), N,N,N’,N’-tetra methyl ethylenediamine (TEMED), bovine serum albumin and Bradford reagents were purchased from BioRad. Propan-2-ol, ethanol, sorbitol, HCl, MgCl2, MnCl2 and (NH4)2SO4 were from Prolabo. Glycerol was obtained from Acros organique.
5
Stress Tolerance Assays for Bacteria
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NO, HClO, DOC, CHO, SDS, Triton X-100 and diamide stress assays were performed in TY by using the serial dilution protocol as indicated in the O2-tolerance section. Dilutions of a suspension at an OD600nm of 0.5 were plated on TY plate and in TY plate containing either 750 μM of DEA NONOate (Sigma-Aldrich), 0.1% of NaClO (Sigma-Aldrich), 0.03% (725 μM) of DOC (Sigma-Aldrich), 0.4% (9.3 mM) of CHO (Sigma-Aldrich), 0.1% (2.2 mM) of GlyDOC (Sigma-Aldrich), 0.01% of Triton X-100 (Merck), 0.003% of SDS (Euromedex) or 0.2 mM of diamide (Sigma-Aldrich). When indicated, DTT (Sigma-Aldrich) was added at 0.1%. For each plate, the last dilution allowing growth was recorded after incubation at 37°C for 24 h (NO, HClO, diamide) or 48 h (DOC, CHO, SDS, triton). The survival was evaluated by doing the ratio of the CFUs in presence of the stress on the CFUs on the control plate. For H2O2, overday cultures were used to prepare two bacterial suspensions per strain at an OD600nm of 0.5 in glycyl-glycine buffer (glycylglycine 50 mM, glucose 0.2%, pH 8). H2O2 (Honeywell) was added in one of the suspensions to a final concentration of 250 μM. After 30 minutes, the suspensions were serially diluted and plated on TY plate and survival was calculated as mentioned above. Experiments were performed in 5 biological replicates.
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