N2A cells were co-transfected with pcDNA3-Htt(Q)25-eYFP or pcDNA3-Htt(Q)109-eYFP and either pcDNA3 vector or pcDNA3.1-FKBP12–V5. After 48 hours, the transfected cells were harvested in 500 μl of ice-cooled PBS buffer containing protease inhibitor cocktail (Roche) and Benzonase Nuclease (Merck Millipore), and sonicated on ice for 1 min. Extracts were centrifuged at 14 K rpm for 30 min at 4 °C, and concentrations were determined using the BCA assay. Samples containing 120 μg of total proteins in a volume of 500 μl were filtered with a 0.22 μm filter (Millipore) and fractionated with a Superdex 200 10/300 column (GE Healthcare) using a flow rate of 0.3 ml/min. Each fraction (1 ml volume/fraction) was collected and subjected to Western blot and slot blot analysis. Htt oligomeric species in each fraction were quantified by densitometry (Image J). The percentage of Htt species was calculated by dividing the density of each fraction by the summed density of every fraction (fractions 7–18). For slot-blotting analysis, the collected fractions were applied to a 0.45 μm nitrocellulose membrane (Schleicher & Schuell) and probed with an A11 antibody (1:1000, Invitrogen) overnight at 4 °C, followed by incubation with HRP-conjugated anti-rabbit secondary antibody (1:7500; Jackson ImmunoResearch) for 1 hour at room temperature. Blots were detected using an ECL chemiluminescent kit.
0.45 μm nitrocellulose membrane
Manufactured by Cytiva
Sourced in United Kingdom, United States, Germany
The 0.45 μm nitrocellulose membrane is a laboratory filtration product designed for size-based separation of particles, molecules, and cells. It has a nominal pore size of 0.45 micrometers, making it suitable for a variety of filtration applications. The membrane is composed of nitrocellulose material, which is a common choice for biological and chemical analysis techniques.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Roche (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Superdex 200 10 300 column, by GE Healthcare (1 mentions)
Benzonase nuclease, by Merck Group (1 mentions)
A11 antibody, by Thermo Fisher Scientific (1 mentions)
0.22 μm filter, by Merck Group (1 mentions)
Hrp conjugated anti rabbit secondary antibody, by Jackson ImmunoResearch (1 mentions)
Bio dot apparatus, by Bio-Rad (1 mentions)
Ab110273, by Abcam (1 mentions)
Ab110267, by Abcam (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Amersham ecl prime detection reagent, by GE Healthcare (1 mentions)
Amersham ecl prime, by Cytiva (1 mentions)
Enhanced chemiluminescence western blot substrate, by Bio-Rad (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Phosphostop, by Roche (1 mentions)
Odyssey clx imager, by LI COR (1 mentions)
Nanodrop one, by Thermo Fisher Scientific (1 mentions)
Dnazol, by Thermo Fisher Scientific (1 mentions)
28 protocols using 0.45 μm nitrocellulose membrane
1
Characterization of Huntingtin Oligomers
2
Fly Protein Extraction and Western Blot
3
Western Blot Analysis of ABCG2 Expression
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Cells were lysed in whole lysis buffer (1% Nonidet P-40, 150 mM NaCl, 50 mM Tris, pH 7.5–8.0, 0.02% sodium azide, 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 100 μg/mL PMSF, 1 μg/mL aprotinin), and protein concentrations were determined using the Bio-Rad protein assay. Equal amounts of cell lysates were subjected to SDS-polyacrylamide gel electrophoresis, and then transferred to a 0.45 μm nitrocellulose membrane (Amersham, Sweden). Membranes were blocked with 5% skimmed milk-PBS/0.1%. Tween 20 for an hour prior to an overnight incubation at 4 °C with primary antibody (anti-ABCG2, 1:500 and β-tubulin, 1:200, all diluted in 5% skimmed milk in PBS/0.1% Tween 20). The membrane was then incubated with HRP-conjugated secondary antibody. Following successive washes, membranes were developed using an enhanced ECL detection system. β-tubulin was used as an internal control. Anti-ATP5A antibody (ab110273), abcam and Anti-Cytochrome C Oxidase subunit VIc antibody (ab110267), a cam.
4
Immunoblotting for Protein Detection
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Immunological detection by immunoblotting was done according to the method of Bollag and Edelstein [32 ]. Samples were subjected to SDS-PAGE as previously described and transferred to a 0.45 μm nitrocellulose membrane (Amersham Biosciences, UK) at a constant current of 100 mA for 1 h at 4°C. Membranes were then rinsed with TBS, blocked with 3% (w/v) bovine serum albumin ON at 20°C, and washed three times with TBS for 10 min. Blots were then probed with an a-His antibody (Santa Cruz Biotechnology Inc, Dallas, TX, USA) or polyclonal antibodies raised against recombinant A thaliana SEX4 [33 (link)] affinity-purified against OsttaDSP as described by Plaxton [38 (link)]. After primary antibody incubation, membranes were washed three times in TBS for 10 min and then incubated with 1:30000 a-rabbit IgG alkaline phosphatase conjugated or a-rabbit IgG peroxidase conjugate (for O. tauri crude extracts analysis) secondary antibodies (Sigma-Aldrich) for 1 h at 22°C. Blots were then carefully washed before detection with BCIP-NBT or Amersham ECL Prime detection reagent (GE Healthcare bio-Sciences) as outlined by the manufacturer.
5
Western Blot Analysis of Cellular Proteins
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Protein extracts were applied on 15% SDS-PAGE gels and transferred to a 0.45 μm nitrocellulose membrane (Amersham). Membrane was blocked in 5% non-fat dry milk in TBS-T [TBS with 0.05% Tween (Sigma-Aldrich)] for 1 h, prior to overnight incubation at 4 °C with primary antibodies: rabbit anti-hTTR (A0002, DAKO, 1:1,000), mouse anti-α-Tubulin (T5168, clone B512, Sigma-Aldrich, 1:100,000), mouse anti-GAPDH (G-9, sc365062, Santacruz, 1:500), mouse anti-Rho1 (P1D9, Developmental Studies Hybridoma Bank, 1:250), mouse anti-Rac1 (23A8, ab33186 Abcam, 1:2000) in blocking buffer. Incubation with horseradish peroxidase-labeled secondary antibodies was performed for 1 h at room temperature in blocking buffer. Blots were developed using the enhanced chemiluminescence western blot substrate (Bio-Rad).
6
Western Blot Analysis of Protein Extracts
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Cells were lysed in ice for 20 min using RIPA buffer, added with phosphatase (Phosphostop, Roche, #04,906,837,001) and protease (Complete, #11,836,153,001) inhibitor cocktails, and 1 mM PMSF. Lysates were then centrifuged at 13,000 rpm for 20 min and the supernatants stored at -80 °C. Proteins were quantified through Pierce BCA Protein Assay Kit (Thermofisher, #23,225) and optical density evaluated by Spark Multimode microplate reader. An equal amount of proteins (20 µg) was run on NuPAGE 4–12% pre-casted minigels (Invitrogen, #NP0321BOX) and proteins were transferred to 0.45 μm nitrocellulose membrane (Amersham, #10,600,002). Nonspecific binding sites were blocked in PBS containing 0.1% Tween 20 (Sigma, #P2287-500ML) and 5% BSA (Sigma, #A7906-100G).
Membranes were incubated overnight at 4 °C with the different antibodies listed inSupplementary Material and Methods . After incubation, membranes were washed three times, for 10 min each, in PBS containing 0.1% Tween 20, and then incubated for 30 min at room temperature with the following secondary antibodies: Rabbit anti-goat (Invitrogen, #811,620) and Donkey anti-rabbit (Invitrogen, #A16035). After washing, blots were developed with Clarity Western ECL Substrate (Biorad, #1,705,061) and images were acquired using Chemidoc XRS system (Biorad). Quantification was done with ImageJ 64 software.
Membranes were incubated overnight at 4 °C with the different antibodies listed in
7
Quantifying TOP1-DNA Covalent Complexes
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RADAR protocol was performed as described (48 ). Cells (4.5 × 105) were seeded in six-well plates and then treated according to the experimental scheme. After CPT treatment, the medium was removed, and cells were lysed with 1 ml of DNAzol (10503027, Thermo Fisher Scientific) and collected by scraping. Genomic DNA and DNA-protein covalent complexes were precipitated at −20°C by addition of 0.5 volume of 100% cold ethanol, recovered by centrifugation at 10000 rpm at 4°C, washed twice in 70% ethanol, and resuspended in 300 μl of freshly prepared 8 mM NaOH after air drying for 3 min. The DNA content was measured with a NanoDrop One instrument (Thermo Fisher Scientific), normalized in 1 ml of tris-buffered saline buffer at 10 ng/μl, and measured again before starting with the slot blotting procedure. DNA was then deposited onto a 0.45-μm nitrocellulose membrane (Amersham) using a slot blotting apparatus (1706542, Bio-Rad). TOP1-ccs were detected by overnight incubation with primary rabbit anti-TOP1 antibody (1:2000; ab28432, Abcam, RRID:AB_778545) and secondary IRDye 800CW goat anti-mouse immunoglobulin G (926-32210, LI-COR). Images were acquired with Odyssey CLx Imager (LI-COR), and TOP1-ccs were quantified by ImageStudio Software (RRID: SCR_013715) and are expressed as fold change normalized on the untreated WT sample.
8
Sema7A-Induced FAK Signaling in GSCs
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GSC were seeded at 1 × 104/cm2 in complete Neurobasal-A medium. After 24 h, cells were treated with Human Recombinant SEMA7A Fc Chimera (R&D System, Minneapolis, Minnesota) for 2, 5 or 10 min at 10 or 100 ng/mL or untreated.
For treatments with GASC-exosomes, GSCs were exposed to 10 µg/mL of exosomes for 5, 10 and 30 min. For treatments with anti-integrin beta1 blocking antibody, GSC were treated as detailed inAppendix A .
Cells were washed in cold PBS, immediately harvested in RIPA buffer, at 4 °C, and proteins were extracted as previously described. Twenty micrograms of proteins was resolved on 10% SDS/PAGE, and then transferred and immobilised on a 0.45 μm nitrocellulose membrane, (Amersham).
The membranes were incubated with rabbit-anti human to FAK antibody, rabbit-anti human to phospho-FAK (Tyr-397) antibody (Cell Signalling Technology, Danvers, Massachusetts) and with rabbit polyclonal to beta-Actin. Levels of FAK and p-FAK were evaluated by densiometric analysis, using ImageJ. IOD (Integrated Optical Density) was analysed for each condition and Fold Change of p-FAK was calculated vs. untreated (Ctrl) cells, after normalisation on β -actin expression.
For treatments with GASC-exosomes, GSCs were exposed to 10 µg/mL of exosomes for 5, 10 and 30 min. For treatments with anti-integrin beta1 blocking antibody, GSC were treated as detailed in
Cells were washed in cold PBS, immediately harvested in RIPA buffer, at 4 °C, and proteins were extracted as previously described. Twenty micrograms of proteins was resolved on 10% SDS/PAGE, and then transferred and immobilised on a 0.45 μm nitrocellulose membrane, (Amersham).
The membranes were incubated with rabbit-anti human to FAK antibody, rabbit-anti human to phospho-FAK (Tyr-397) antibody (Cell Signalling Technology, Danvers, Massachusetts) and with rabbit polyclonal to beta-Actin. Levels of FAK and p-FAK were evaluated by densiometric analysis, using ImageJ. IOD (Integrated Optical Density) was analysed for each condition and Fold Change of p-FAK was calculated vs. untreated (Ctrl) cells, after normalisation on β -actin expression.
9
Exosomal Protein Characterization by Western Blot
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Whole-cell and exosomes extract proteins were obtained by lysis in RIPA buffer. Thirty micrograms of proteins was resolved on SDS-PAGE, transferred and immobilised on a 0.45 μm nitrocellulose membrane (Amersham, London, UK).
Membranes were incubated overnight at 4 °C, with goat polyclonal to CD63 (LSBio, Seattle, Washington) mouse monoclonal to CD9 (Clone Ts9) (Thermo Fisher, Walthman, Massachussetts) mouse monoclonal to TSG101 (Clone 4A10) (Abcam, Cambridge, UK), rabbit polyclonal to Calnexin (Abcam rabbit polyclonal to SEMA7A (Novus Biologicals, Colorado, US) and rabbit polyclonal to beta-Actin (Sigma-Aldrich, Italy). Primary antibodies were detected using horseradish peroxidase-linked secondary antibodies (DAKO, Cambridge, UK) and visualised using the enhanced chemiluminescent detection system (SuperSignal West Dura Extended Duration Substrate, Thermo Scientific, Walthman, Massachussetts); seeAppendix A .
Membranes were incubated overnight at 4 °C, with goat polyclonal to CD63 (LSBio, Seattle, Washington) mouse monoclonal to CD9 (Clone Ts9) (Thermo Fisher, Walthman, Massachussetts) mouse monoclonal to TSG101 (Clone 4A10) (Abcam, Cambridge, UK), rabbit polyclonal to Calnexin (Abcam rabbit polyclonal to SEMA7A (Novus Biologicals, Colorado, US) and rabbit polyclonal to beta-Actin (Sigma-Aldrich, Italy). Primary antibodies were detected using horseradish peroxidase-linked secondary antibodies (DAKO, Cambridge, UK) and visualised using the enhanced chemiluminescent detection system (SuperSignal West Dura Extended Duration Substrate, Thermo Scientific, Walthman, Massachussetts); see
10
Western Blot Analysis of Plant Proteins
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Equal weight samples of N. benthamiana leaves were ground to powder in liquid nitrogen, then resuspended in lysis buffer [100 mM Tris–HCl (pH 8.8), 6% sodium dodecyl sulfate (SDS), 2% β-mercaptoethanol] and centrifuged at 4°C for 12,000 rpm. Total proteins in the supernatants were separated in 12% SDS-PAGE (TGX Stain-Free FastCast Acrylamide Kit, Bio-Rad) and transferred onto 0.45 μm nitrocellulose membrane (Amersham Biosciences, United Kingdom) using semi-dry electroblotting, then incubated with relevant primary antibodies including anti-NbLHCB3 (YouLong Biotech, China), anti-TuMV CP (NEOGEN, United Kingdom), anti-GFP (Transgen Biotech, China) and anti-c-Myc (Gene Tex, Irvine, CA, United States). Secondary antibodies were AP-linked anti-rabbit or anti-mouse (Sigma-Aldrich, St Louis, MO, United States). The antigen-antibody complex was visualized by adding nitrotetrazolium blue chloride/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) (Sigma-Aldrich) at room temperature for a few minutes and results were captured with a Canon digital camera.
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