Patient-derived fibroblasts were grown on 15-cm plates and were washed twice with ice-cold PBS buffer (Thermo Fisher Scientific) prior to lysis. The cells were lysed by addition of lysis buffer (50 mm Tris-HCl, pH 7.4, 1 mm EGTA, 1 mm EDTA, 1% Triton X-100, 1 mm Na3VO4, 50 mm NaF, 5 mm pyrophosphate, 0.27 m sucrose) supplemented with 1 μm GlcNAcstatin-G, 1 μm β-mercaptoethanol, and protease inhibitor mixture (1 mm benzamidine, 0.2 mm PMSF, 5 mm leupeptin). The lysate was transferred into an Eppendorf and clarified by centrifugation at 4 °C (1200 × g for 15 min). Lysate proteins were resolved by SDS-PAGE (4–12% acrylamide; Thermo Fisher Scientific) and transferred onto nitrocellulose membranes (GE Healthcare). Analysis of O-GlcNAcylated fibroblast proteins by the far Western method was performed as described previously (32 (link)). Briefly, soluble cell lysates were prepared, resolved by SDS-PAGE (3–8% Tris acetate; Thermo Fisher Scientific) and transferred onto nitrocellulose membranes (GE Healthcare) as described above, but with lysis buffer lacking GlcNAcstatin-G.
Sds page
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SDS-PAGE is a laboratory technique used for the separation and analysis of proteins based on their molecular weight. It is a type of gel electrophoresis that utilizes the detergent sodium dodecyl sulfate (SDS) to denature proteins and give them a uniform negative charge, allowing them to be separated solely by their molecular weight as they migrate through a polyacrylamide gel matrix under an applied electrical field.
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Lab products found in correlation
Pvdf membrane, by Merck Group (45 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (31 mentions)
Ripa buffer, by Thermo Fisher Scientific (21 mentions)
Ripa buffer, by Merck Group (19 mentions)
Protease inhibitor cocktail, by Roche (16 mentions)
Polyvinylidene fluoride membrane, by Merck Group (14 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (13 mentions)
Odyssey infrared imaging system, by LI COR (12 mentions)
Pvdf membrane, by Thermo Fisher Scientific (12 mentions)
Nitrocellulose membrane, by Bio-Rad (11 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (11 mentions)
Pvdf membrane, by Bio-Rad (10 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (10 mentions)
Nitrocellulose membrane, by Cytiva (9 mentions)
β actin, by Merck Group (9 mentions)
Nitrocellulose membrane, by GE Healthcare (9 mentions)
Ecl plus, by GE Healthcare (9 mentions)
β actin, by Cell Signaling Technology (9 mentions)
Iblot, by Thermo Fisher Scientific (8 mentions)
Gapdh, by Cell Signaling Technology (7 mentions)
402 protocols using sds page
1
Analysis of O-GlcNAcylated Fibroblast Proteins
2
Western Blot Analysis of Adipocyte Proteins
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Cells were lysed using RIPA Buffer (Thermo Fisher Scientific) supplemented with protease inhibitor cocktail set I (EMD Millipore, Burlington, MA, USA). Protein samples were separated on 4–20% SDS-PAGE (Thermo Fisher Scientific) and transferred for 90 min at 40 V onto PVDF membranes (EMD Millipore). Membranes were blocked using Odyssey blocking buffer (Licor). Membranes were incubated with primary antibodies at 4 °C overnight then washed with PBS-Tween and incubated with secondary antibodies for 1 h at room temperature. Western blots were imaged and quantitated using the Odyssey infrared imaging system. Western blot analysis was carried out using the following antibodies: UCP1 (Abcam ab155117, 1:500), FABP4 (Abcam ab66682, 1:1000), and β-actin (Santa Cruz sc-47778, 1:1000).
3
Western Blot Validation of Knockdown Experiments
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For verification of knockdown experiments, cells were lysed in RIPA buffer 48 h after transfection. Extracted proteins were separated by 4–20% SDS-PAGE (Thermo Fisher Scientific), transferred to PVDF membranes (Bio-Rad Laboratories), incubated at 75 V for 2 h, and blocked for 1 h with 5% nonfat milk (Bio-Rad Laboratories) or BSA (Sigma-Aldrich). The PVDF membranes were incubated overnight at 4°C with appropriate antibodies: anti-ARF1 (dilution 1:1,000; ab108347; Abcam); anti-ARF6 (dilution 1:1,000; ab77581; Abcam); anti-ARNO (dilution 1:1,000; ab56510; Abcam); anti–cytohesin 1 (dilution 1:500; MABT14; EMD Millipore); anti–α-tubulin (dilution 1:3,000; T6199; Sigma-Aldrich); anti-βCOP (dilution 1:1,000; ab2899; Abcam); anti-HA (dilution 1:1,000; 2367; Cell Signaling Technology); anti-Cdc42 (dilution 1:1,000; 2462; Cell Signaling Technology); anti-RhoA (dilution 1:1,000; sc-418; Santa Cruz Biotechnology, Inc.); anti-Rac1 (dilution 1:1,000; 610650; BD); and anti–NM myosin-IIA (dilution 1:1,000; M8064; Sigma-Aldrich).
After three washes (10 min each), appropriate secondary antibodies conjugated with HRP (Bio-Rad Laboratories) were incubated with the membrane for 1 h, washed three times (15 min at RT), and detected by ECL Western blotting substratum (Thermo Fisher Scientific) using CL-Xposure film (Thermo Fisher Scientific).
After three washes (10 min each), appropriate secondary antibodies conjugated with HRP (Bio-Rad Laboratories) were incubated with the membrane for 1 h, washed three times (15 min at RT), and detected by ECL Western blotting substratum (Thermo Fisher Scientific) using CL-Xposure film (Thermo Fisher Scientific).
4
Protein Extraction and Western Blotting of C. elegans
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Total protein was extracted from 50 handpicked Day 1 adult animals grown on OP50 bacteria at 20°C. Animals were washed thoroughly in M9 buffer and centrifuged, and the pellets were lysed in 10 μl of 6x SDS sample buffer. The entire extract was separated by 4–20% SDS-PAGE (Thermo Fisher Scientific, Waltham, MA) and transferred to a PVDF membrane (Millipore, Hayward, CA). Immunoblotting was performed using primary anti-GFP (diluted 1:1000; Santa Cruz Biotechnology, Dallas, TX), anti-mCherry (diluted 1:500; Clontech, Mountain View, CA), and anti-LGG-1 (diluted 1:1000; sample kindly obtained from Abgent, San Diego, CA) antibodies and secondary horseradish peroxidase-conjugated goat anti-mouse (diluted 1:2000; Santa Cruz Biotechnology, Dallas, TX) or goat anti-rabbit (1:2000; Cell Signaling Technology, Danvers, MA) secondary antibodies. Immunoblots were developed using enhanced chemiluminescent reagent (Thermo Fisher Scientific, Waltham, MA).
5
Western Blot Analysis of Gremlin-1
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Protein extracts were prepared with RIPA lysis buffer solution (Sigma-Aldrich, St. Louis, MO) supplemented with 1× protease inhibitor cocktail (Roche) and 1 mM sodium orthovanadate. Protein lysates were boiled for 5 Min in SDS-sample buffer before being applied into a 4–20% SDS-PAGE (Thermo Fisher Scientific, Rockford, IL). After electrotransfer to nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) and blocking in TBS-T buffer containing 5% non-fat milk for 1 h, blots were incubated with Gremlin-1 primary antibody (Santa Cruz, Santa Cruz, CA) overnight. The subsequent incubation with the peroxidase-conjugated secondary antibody was followed by detection using ECL Western blotting detection reagents (GE Healthcare) and the FusionSL 4 3500 WL detection system (Vilber Lourmat, Sud Torcy, France). Membranes were incubated with Restore Western Blot Stripping Buffer (Thermo Fisher Scientific) followed by incubation with an α-Tubulin antibody (Sigma-Aldrich) as reference protein. Quantification of protein amount was determined using Bio-1D software (Vilber Lourmat).
6
Western Blot Analysis of GLI1 and β-Actin
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Proteins of MV4-11, MOLM-13, and OCI-AML3 cells were extracted using the trichloroacetic acid method. Protein extracts were applied to a 4–20% SDS-PAGE (Thermo Fisher Scientific, Rockford, IL) followed by electrotransfer to nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). Blots were incubated with either rabbit anti-human GLI1 (C68H3, Cell Signaling Technology, RRID:AB_1903989) or mouse anti-human β-Actin (sc-47778, Santa Cruz Biotechnology, RRID:AB_2714189) at 4 °C overnight. The subsequent incubation with the peroxidase-conjugated secondary antibodies (anti-mouse IgG, NXA931, GE healthcare, RRID:AB_772209 and anti-rabbit IgG, 7074S, Cell Signaling Technology, RRID:AB_2099233) was followed by detection using ECL Western blotting detection reagents (GE Healthcare) and the FusionSL 4 3500 WL detection system (Vilber Lourmat, Sud Torcy, France).
7
Protein Visualization and Analysis
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Proteins were extracted from cells grown to an OD550 of 1.0, separated by 4–12% SDS-PAGE (Invitrogen), transferred to nitrocellulose membrane, and analyzed using primary antibodies to GFP (1:1,000; Invitrogen), Dpm1p (1:500; Thermo Fisher Scientific), Pex3p (1:1,000; provided by R. Erdmann, Ruhr University, Bochum, Germany), and Porin (1:1,000; Invitrogen). Proteins were visualized using appropriate IRDye secondary antibody (LI-COR Biosciences; 1:10,000) followed by detection using the Odyssey system.
8
Immunoprecipitation and Western Blot Analysis
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Cultured cells or brain tissues were homogenized in NP-40,1 × PBS or RIPA buffer with 1× protease inhibitor as previously described [57 (link)]. For immunoprecipitation, the lysates were centrifuged at 800 × g for 5 min, and supernatants were incubated with antibody and Protein A beads (Sigma-Aldrich) at 4 °C overnight, followed by washing with lysis buffer 4 times. The samples were subjected to 4-12 % SDS-PAGE (Invitrogen) and detected with SuperSignal West Dura Extended Duration Substrate (Thermo). For RNase treatment, pig cortex lysates were incubated with 200 μg/ml RNase A for 30 min at 25 °C before immunoprecipitation.
9
Western Blot Protein Extraction and Analysis
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Cells were washed in PBS and incubated for 30 min at 4 °C in NaDOC lysis buffer [50 mM Tris·HCl (pH 7.4)/150 mM NaCl/5 mM EDTA/0.5% Triton X-100/0.5% sodium deoxycholate] and a mixture of phosphatase (Thermo-Scientific, Waltham, MA, USA) and protease (Roche, Mannheim, Germany) inhibitors. Extracts were centrifuged at 14,000 xg for 15 min. Protein concentrations in the supernatant were measured by using the bicinchoninic acid method (Pierce, Rockford, IL, USA). Protein extracts (15 μg) were resolved by 4–12% SDS-PAGE (Invitrogen) and transferred to nitrocellulose membranes (iBlot, Invitrogen). Membranes were blocked with SEABLOCK blocking buffer (Thermo-Scientific) for 1 h at room temperature and then incubated overnight at 4 °C with primary antibody. Bound antibody was revealed by infrared detection using a secondary antibody coupled to IRDye fluorophores (Li-Cor biosciences, Lincoln, NE, USA). Western blot read out was performed with the Odyssey Infrared Imaging System (Li-Cor biosciences).
10
Protein Expression Analysis Protocol
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Equal amounts of protein were resolved by 4–12% SDS-PAGE (Invitrogen) and transferred to nitrocellulose membranes (GE Healthcare Bio-sciences, UK). The membranes were blocked with 5% skim milk-blocking buffer for 1 h before incubation overnight at 4°C with primary antibodies. The membranes were then washed three times and incubated with horseradish peroxidase-conjugated secondary antibodies in blocking buffer for 1 h. After successive washes, the membranes were developed using SuperSignal West Pico Chemiluminescent kit (Thermo Fisher Scientific, USA).
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