Cells were transfected with polyethylenimine as previously described [42 (link)]. Cells were lysed in a cold, near-neutral pH solution containing 150 mM NaCl, 50 mM Tris pH 7.5 or 50mM sodium acetate pH5.3, 1% Triton X-100, 0.1% deoxycholic acid, 1X protease inhibitors (Roche). After centrifugation at 14,000 xg, for 15 minutes, at 4°C, supernatants were transferred to clean tubes on ice, to which rabbit anti-PGRN antibody-conjugated Affi-Gel 15 (Bio-Rad Laboratories), Myc-Trap or GFP-Trap beads were added, then rocked for 3–4 hours at 4°C. Samples were run on 12% polyacrylamide gels or 4–12% Bis-Tris gels (Invitrogen), then transferred to Immobilon-FL polyvinylidene fluoride membranes (Millipore Corporation) or nitrocellulose membranes (Millipore Corporation). Membranes were blocked with either 5% non-fat milk in PBS or Odyssey Blocking Buffer (LI-COR Biosciences) for 1 hour then washed with Tris-buffered saline with 0.1% Tween-20 (TBST) 3x for 5 minutes each. Membranes were incubated with primary antibodies, rocking overnight at 4°C, then washed as above, incubated with secondary antibodies for 2 hours at room temperature, then washed again. Membranes were scanned using an Odyssey Infrared Imaging System (LI-COR Biosciences). Densitometry was performed with Image Studio (LI-COR Biosciences).
Immobilon fl polyvinylidene fluoride membrane
Manufactured by Merck Group
Immobilon-FL polyvinylidene fluoride membrane is a laboratory product used for protein and nucleic acid immobilization and transfer. It is a thin, hydrophobic polymer membrane that provides a surface for binding biomolecules. The membrane can be used in various analytical techniques, such as western blotting, dot blotting, and southern blotting.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey blocking buffer, by LI COR (2 mentions)
Nupage gel, by Thermo Fisher Scientific (2 mentions)
Odyssey clx scanner, by LI COR (2 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Image studio, by LI COR (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Anti rabbit igg conjugated to hrp, by GE Healthcare (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Dextran coated charcoal, by Merck Group (1 mentions)
Fk506, by Cell Signaling Technology (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Sodium orthovanadate, by Merck Group (1 mentions)
Sodium fluoride, by Merck Group (1 mentions)
Criterion blotter, by Bio-Rad (1 mentions)
Lds loading buffer, by Thermo Fisher Scientific (1 mentions)
Bolt sample reducing agent, by Thermo Fisher Scientific (1 mentions)
N per lysis buffer, by Thermo Fisher Scientific (1 mentions)
8 protocols using immobilon fl polyvinylidene fluoride membrane
1
Progranulin Immunoprecipitation and Western Blot
2
Immunoblot Analysis of NLRC4 in Macrophages
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Macrophages were grown in tissue culture–treated 24-well plates, medium was removed, and cells were lysed in plate in radioimmunoprecipitation assay buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, and 1× Roche protease inhibitor tablet [no EDTA], pH 8.0) for 20 min at 4°C. Lysates were clarified by centrifugation at 16,000 g for 10 min at 4°C, and supernatants were separated by 4–12% SDS-PAGE in morpholinoethanesulfonic acid buffer (Invitrogen). Proteins were transferred to Immobilon-FL polyvinylidene fluoride membranes (Millipore). Membranes were blocked with 5% milk. Anti-NLRC4 antibody (gift of S. Mariathasan and V. Dixit, Genentech, South San Francisco, CA) was detected with a secondary anti-rabbit IgG conjugated to HRP (GE Healthcare).
3
Signaling Pathway Inhibition Assay
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Dexamethasone (Dex), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), dulbecco’s modified eagle’s medium (DMEM), powdered medium, Tris, ethylenediaminetetraacetic acid (EDTA), phosphate buffered saline (PBS), sodium orthovanadate, sodium fluoride, protease inhibitor cocktail, dextran coated charcoal, and sodium chloride were all obtained from Sigma (St. Louis, MO). Iron-supplemented bovine calf serum was from Hyclone Laboratories Inc. (Logan, UT). Immobilon-FL polyvinylidenefluoride membrane was obtained from Millipore Corporation (Bedford, MA). Puromycin was from Fisher Scientific (Pittsburgh, PA). FK506 was from Cell Signaling Technology, Inc. (Boston, MA). VX-853 (as timcodar dimesylate) was a gift from Dr. Bruce Gold (Oregon Health and Science University).
4
Western Blot Protocol: Protein Detection
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Cells were lysed in N-PER lysis buffer (ThermoFisher) supplemented with 1× Halt Protease and Phosphatase Inhibitor Cocktail (ThermoFisher). Lysates were reduced in Bolt Sample Reducing Agent (ThermoFisher) and LDS loading buffer (ThermoFisher) at 75 °C for 5 min. The samples were electrophoresed on a 4–12% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis gel (ThermoFisher) and electroblotted onto a 0.45-μm Immobilon-FL polyvinylidene fluoride membrane (Millipore) by using the Criterion Blotter (Bio-Rad). The membranes were blocked with a 1:2 dilution of Odyssey Blocking Buffer (LI-COR) in phosphate-buffered saline (PBS) for 1 h at room temperature then incubated with primary antibodies overnight at 4 °C. Odyssey IRDye secondary antibodies (LI-COR) were used for the detection on an Odyssey CLx scanner (LI-COR).
5
Quantifying Polycomb Protein Levels
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To quantify protein level, cells were collected and lysed with buffer (20 mM Tris-HCl, pH 7.4, 2.0% NP-40, 1.0% Triton X-100, 500 mM NaCl, 0.25 mM EDTA, 0.1 mM Na3VO4, 0.1 mM PMSF, and protease inhibitors [P8340; Sigma–Aldrich]). After centrifugation, the protein concentration was quantified and normalized to the same concentration. Proteins were separated using SDS–PAGE, transferred to 0.45-μm Immobilon-FL polyvinylidene fluoride membrane (Millipore, Darmstadt, Germany), and probed with anti-Cbx2 (ab80044; Abcam, Cambridge, MA), anti-Phc1 (6-1-3; Active Motif, Carlsbad, CA), anti-Ring1b (D139-3; MBL, Woburn, MA), and anti-Mel18 (sc-10744; Santa Cruz Biotechnology, Dallas, TX). Proteins were detected using ECL Plus detection reagents (GE Healthcare, Pittsburgh, PA). Membranes were imaged using a ChemiDoc XRS system (Bio-Rad, Hercules, CA).
6
Immunoblotting Using Odyssey IR Imaging
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Western blot analyses were performed as described elsewhere (33 (link)). In some immunoblot analyses, samples were transferred onto an Immobilon-FL polyvinylidene fluoride membrane (Millipore Corporation) and probed with primary and IRDye 800CW– or IRDye 680–conjugated secondary antibodies (LI-COR Biosciences). Immunoreactive bands were detected using the Odyssey IR Imaging System (LI-COR Biosciences).
7
Quantitative Immunoblotting for Protein Analysis
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For immunoblot analyses, cell lysates were loaded and run on a 4–12% NuPAGE gel (Invitrogen) and transferred to an Immobilon-FL polyvinylidene fluoride membrane (IPFL00010, Millipore) before immunoblotting (Di Fiore and Pines, 2010 (link)). Primary antibodies were used at the indicated concentrations: anti-MAD1 (clone 9B10, 1:400, mouse, Sigma-Aldrich, 2 mg/ml), anti-Flag (M2, F3165, 1:4,000, mouse, Sigma-Aldrich), anti-Mad2 (A300-301A; 1:1,000, rabbit, Bethyl Laboratories), and anti-cyclin B1 (GNSI, 1:500, PharMingen). IRDye800CW donkey anti-mouse (926–32212, LI-COR), IRDye800CW donkey anti-rabbit (926–32213, LI-COR), IRDye680CW donkey anti-mouse (926–68072, LI-COR), and IRDye680CW donkey anti-rabbit (926–68073, LI-COR) secondary antibodies were all used at 1:10,000. Quantitative immunoblotting was performed on a LI-COR Odyssey CLx scanner according to the manufacturer’s instructions (LI-COR Biosciences).
8
Cdk1 Immunodepletion and Immunoblotting
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For Cdk1 immunodepletion (Fig. 2 For all IPs, beads were washed 5 times with IP buffer and then incubated 5 minutes at 65°C in 30 ul 2X Sample Loading Buffer, prior to SDS-PAGE.
Immunoblotting 40 ug of RPE cell lysates were separated through SDS-PAGE on a 4-12% NuPAGE gel (Invitrogen) and transferred to an Immobilon-FL polyvinylidene fluoride membrane (IPFL00010, Millipore). The membrane was blocked with 5% Milk, 0.1 % Tween, PBS and incubated overnight with primary antibodies at 4°C in 2.5% Milk, 0.1 % Tween, PBS. The following day the membrane was washed with 0.1% Tween PBS and incubated with secondary antibodies in 2.5% Milk, 0.1 % Tween, PBS for 1h at RT.
Primary antibodies were used at the indicated concentrations: and anti-CCNB1 (1:1000, SantaCruz -GSN1, sc-245), anti-Cdk1 (1:1000, BD Biosciences -610037), anti-Cdk2
(1:1000, 78B2 -Cell Signalling), anti-Tubulin (1:3000, ab6046 -Abcam). IRDye800CW
Immunoblotting 40 ug of RPE cell lysates were separated through SDS-PAGE on a 4-12% NuPAGE gel (Invitrogen) and transferred to an Immobilon-FL polyvinylidene fluoride membrane (IPFL00010, Millipore). The membrane was blocked with 5% Milk, 0.1 % Tween, PBS and incubated overnight with primary antibodies at 4°C in 2.5% Milk, 0.1 % Tween, PBS. The following day the membrane was washed with 0.1% Tween PBS and incubated with secondary antibodies in 2.5% Milk, 0.1 % Tween, PBS for 1h at RT.
Primary antibodies were used at the indicated concentrations: and anti-CCNB1 (1:1000, SantaCruz -GSN1, sc-245), anti-Cdk1 (1:1000, BD Biosciences -610037), anti-Cdk2
(1:1000, 78B2 -Cell Signalling), anti-Tubulin (1:3000, ab6046 -Abcam). IRDye800CW
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