The filter-binding assay was performed as described previously (Rio 2012 (link)). Of note, 62.4 pmol of purified RNA was 5′-end labeled with 5 µCi [γ-32P] ATP by T4 polynucleotide kinase (New England Biolabs) in nuclease-free buffer at 37°C for 1 h. The reaction was stopped by incubating at 70°C for 10 min. Labelled RNA was purified by mini Quick Spin Oligo Columns (Roche). The purified oligonucleotides were then heated at 95°C for 10 min and cooled down for 30 min. Different concentrations of protein were titrated to a constant amount of 50 nM CAG72 RNA in a final volume of 50 µL and incubated at room temperature for 1 h in binding buffer consisting of 10 mM Tris 7.5, 50 mM KCl, 750 µM MgCl2, 100 µM EDTA, 5% glycerol, 600 µM dithiothreitol, 0.1 mg/mL tRNA, and 40 µg/mL BSA. Nitrocellulose (0.45 µm pore size, Bio-Rad) and nylon filter (Roche) were presoaked in wash buffer 10 mM Tris 7.5, 50 mM KCl, and 1 mM dithiothreitol for 2 h. Both filters, Nitrocellulose on top and nylon filter at the bottom, were placed into a dot-blot apparatus (Bio-Rad). The wells were washed once and vacuum was applied before the samples were loaded. The wells were washed eight times with 100 µL washing buffer after samples were loaded. The Nitrocellulose filters were analyzed using auto-radiography to measure the retained radiolabeled RNA on the Nitrocellulose filter.
Nitrocellulose
Manufactured by Bio-Rad
Sourced in United States, Germany, United Kingdom
Nitrocellulose is a membrane material used in various laboratory techniques. It is a porous, chemically inert substrate that allows for the immobilization and transfer of biomolecules, such as proteins and nucleic acids, during analytical procedures.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (12 mentions)
Odyssey blocking buffer, by LI COR (9 mentions)
Protease inhibitor cocktail, by Roche (8 mentions)
Ripa buffer, by Thermo Fisher Scientific (8 mentions)
Odyssey imaging system, by LI COR (7 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (6 mentions)
Bca protein assay, by Thermo Fisher Scientific (6 mentions)
Laemmli sample buffer, by Bio-Rad (5 mentions)
Complete protease inhibitor cocktail, by Roche (4 mentions)
Anti β actin, by Merck Group (4 mentions)
Odyssey infrared imaging system, by LI COR (4 mentions)
Pvdf membrane, by Bio-Rad (4 mentions)
Phos tag, by Fujifilm (4 mentions)
Polyvinylidene difluoride, by Bio-Rad (4 mentions)
β actin, by Merck Group (4 mentions)
Immobilon p, by Merck Group (4 mentions)
Protease inhibitor, by Roche (4 mentions)
Polyvinylidene difluoride, by Merck Group (4 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (4 mentions)
Bp152 5, by Thermo Fisher Scientific (3 mentions)
194 protocols using nitrocellulose
1
Quantifying RNA-Protein Interactions
2
Western Blot Transfer and Visualization
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Samples were first run on an SDS-PAGE gel as above. Then, the gel was transferred onto nitrocellulose (0.45 μM; Bio-Rad) by removing from its case and placing in a sandwich of gel and nitrocellulose surrounded by filter papers in transfer buffer (Tris base [0.025 M], glycine [0.192 M], 1% SDS [pH 8.6], and 20% methanol). The sandwich was then run in an XCell II Blot Module (Invitrogen) at 100 V for 1 hr. After transfer, the nitrocellulose was blocked by placing in TBS (50 mM Tris and 150 mM NaCl [pH 7.6]) + 3% BSA at 4°C, overnight, with shaking.
Following blocking, the anti-His HRP detection antibody was added at RT for 2 hr, at a dilution of 1:5,000 in blocking buffer. This was then washed off with 3 × 5-min washes with TBS + 0.1% Tween20, before finally placing in TBS. Signals were then developed for imaging by adding enhanced chemiluminescence (ECL) reagent (Clarity Western ECL reagent, Bio-Rad). Images were captured using a Gel Doc (Bio-Rad).
Following blocking, the anti-His HRP detection antibody was added at RT for 2 hr, at a dilution of 1:5,000 in blocking buffer. This was then washed off with 3 × 5-min washes with TBS + 0.1% Tween20, before finally placing in TBS. Signals were then developed for imaging by adding enhanced chemiluminescence (ECL) reagent (Clarity Western ECL reagent, Bio-Rad). Images were captured using a Gel Doc (Bio-Rad).
3
Quantitative Western Blotting for Ubiquitin
4
Isolation and Analysis of Mouse Ileal Epithelial Cells
5
Immunoblotting Protocol for Protein Analysis
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Immunoblotting was performed as previously described17 (link). Briefly, cells were lysed in RIPA buffer, and protein lysates were resolved on Novex 4–12% or 10–20% Tris-Glycine gels (Invitrogen, Carlsbad, CA), transferred to nitrocellulose (Bio-Rad, Hercules, CA) and then incubated with primary antibodies (see Table S2 for primary antibodies). All immunoblotting data shown is a representative of at least 3 biological replicates.
6
Comprehensive Protein Extraction and Analysis
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Proteins from 5 × 106 cells were isolated by 15 min extraction in TX100 lysis buffer: 1% Triton X100, 50 mM Tris pH 7.5, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 50 mM β-glycerophosphate supplemented with 50 mM NaF, 1 mM PMSF, 1 mM DTT, 2 mM Na3VO4, 10 μg/mL Leupeptin, 10 µg/mL Aprotinin, 10 μg/mL Pepstatin, 0.25 ng/mL Microcystin LR (EMD Millipore, Billerica, MA, USA). To achieve total solubilization of vimentin fibers, TX100 lysis buffer was supplemented with 8 M urea. Tissue samples were solubilized by sonication in urea containing TX100 lysis buffer. For Western blotting, proteins (100 μg) were separated by SDS-PAGE (Criterion system, Bio-Rad, Hercules, CA, USA) and transferred to nitrocellulose (Bio-Rad). Antibodies used for Western blotting were: mouse anti-vimentin V9 (MS-129-P, Thermo Scientific, (Waltham, MA, USA) mouse anti-GSK-3αβ (368662, EMD Millipore), mouse anti-α-tubulin (T6074, Sigma-Aldrich, St. Louis, MO, USA), mouse anti-GAPDH (ab9484-200, Abcam, Cambridge, MA, USA) and peroxidase-conjugated secondary antibodies (Jackson Laboratories, Bar Harbor, ME, USA).
7
Zebrafish Plasma Protein Analysis
8
Western Blot Analysis of Protein Lysates
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Protein lysates collected for western blot were clarified by centrifugation at 20,800g for 10 min, followed by quantification using the detergent‐compatible protein assay (BioRad, Hercules, CA) with BSA as a standard. Samples were mixed with 5X loading dye (bromophenol blue, 5% SDS, 30% glycerol, 250 mmol/L Tris‐HCl pH 6.8), boiled for 10 min, resolved on 4–12% gradient SDS‐PAGE gels (BioRad), and transferred to nitrocellulose (BioRad). Blots were blocked, incubated overnight at 4°C in primary antibody as per the manufacturer's instructions (Sp1 antibody, Cat#9389, Cell Signaling, Beverly, MA; Sp3 antibody, Cat#sc‐644, Santa Cruz Biotechnology), incubated in secondary antibody (Anti‐rabbit HRP, Cat#111‐035‐144, Jackson ImmunoResearch, West Grove, PA) for 1 h at room temperature, and developed using Immobilon western chemiluminescent HRP substrate (Millipore, Etobicoke, ON). Images of western blots were captured using QuantityOne software on the BioRad ChemiDoc XRS System and quantified by densitometry.
9
Protein Expression Analysis in ALS Mouse Model
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Tibialis anterior muscles from normal and G93A mice were collected and homogenized in protein extraction buffer containing protease inhibitor cocktail (Thermo Scientific, Waltham, MA) using motorized homogenizer (Wheaton, Millville, NJ). Protein concentrations were determined by BCA protein assay (Thermo Scientific). Equal mass protein samples (30 μg) were subjected to SDS‐polyacrylamide gel electrophoresis before transferred to nitrocellulose (Bio‐Rad, Hercules, CA), and immunoblotted with primary antibodies. The antibodies used were as follows: anti‐phospho‐mTOR (ser2448) (Cell Signaling, Danvers, MA, 5536), anti‐mTOR (Cell Signaling, 2983), anti‐lysozyme (Santa Cruz, Dallas, TX, sc‐27958), anti‐caspase‐3 (Cell Signaling, 9665), anti‐Beclin 1 (Santa Cruz, sc‐10086), anti‐Bcl‐xl (Santa Cruz, sc‐8392). All these antibodies were used at a 1:1000 dilution. One antibody, anti‐tubulin (Santa Cruz Biotechnology), was used at a 1:10000 dilution. Results were visualized with ECL reagents (Thermo Scientific). Densitometry evaluation was conducted using ImageJ software (NIH, Bethesda, MD).
10
Immunoblotting of T Cell Lysates
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