0.1 μm nitrocellulose membrane

Manufactured by GE Healthcare

Sourced in United States

The 0.1-μm nitrocellulose membrane is a laboratory filtration product designed for the separation and isolation of particles, macromolecules, and microorganisms from liquid samples. With a pore size of 0.1 microns, this membrane is capable of retaining a wide range of small particles and molecules while allowing the passage of smaller components. The nitrocellulose material provides a stable and inert substrate for various filtration and analytical applications.

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Lab products found in correlation

Bca assay kit, by Thermo Fisher Scientific (2 mentions) Protease inhibitor cocktail, by Roche (2 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions) Bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Ripa buffer, by Thermo Fisher Scientific (2 mentions) Tris glycine gradient gel, by Bio-Rad (1 mentions) δnp63, by BioLegend (1 mentions) Hrp andsupersignal west pico chemiluminescent substrates, by Thermo Fisher Scientific (1 mentions) Ripa lysis buffer, by Abcam (1 mentions) Hrp conjugated streptavidin, by GE Healthcare (1 mentions) Semi dry apparatus, by GE Healthcare (1 mentions) Clarity ecl, by Bio-Rad (1 mentions) Anti tubulin antibody, by Abcam (1 mentions) Chemidoc mp imaging system, by Bio-Rad (1 mentions) Image lab software, by Bio-Rad (1 mentions) Protease inhibitor, by Thermo Fisher Scientific (1 mentions) Trem2, by Cell Signaling Technology (1 mentions) Tissueruptor, by Qiagen (1 mentions) Peroxidase conjugated donkey anti mouse igg antibody, by Jackson ImmunoResearch (1 mentions) Protease inhibitor cocktail, by Merck Group (1 mentions)

7 protocols using 0.1 μm nitrocellulose membrane

Airway Protein Expression and Ubiquitin Analysis

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Airway cultures were homogenized in RIPA lysis buffer (Abcam; Cambridge,
MA) supplemented with a protease inhibitor cocktail (Roche; Indianapolis, IN).
Following centrifugation at 12,000 rpm for 20 minutes at 4 °C, soluble
supernatant fractions were collected for total protein and western blot
analysis. Total protein concentrations were determined by BCA assay kit (Thermo
Scientific, Waltham, MA). Ten micrograms (μg) total protein were resolved
in pre-casted 4-15% gradient Tris-Glycine gel (Bio-Rad, Hercules, CA), and
immunoblotted for Krt5 (1:5000; Biolegend), ΔNp63 (1:1000; Biolegend),
and K48-ubiquitin (1:1000, R&D Systems). Beta-actin and GAPDH served as
loading controls and for densitometry analysis normalization. Gels were
transferred to 0.1 μm nitrocellulose membrane (GE Healthcare). HRP and
SuperSignal West Pico chemiluminescent substrates (Thermo Scientific) were used
to detect protein signal intensity.

McGraw M.D., Kim S.Y., Reed C., Hernady E., Rahman I., Mariani T.J, & Finkelstein J.N. (2020). Airway basal cell injury after acute diacetyl (2,3-butanedione) vapor exposure. Toxicology letters, 325, 25-33.

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Western Blot Analysis of Crp1 Proteins

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Samples (1/20 vol of fractions containing proteins extracted from the small intestinal tissue) were resolved by AU-PAGE at 150 V and transferred to 0.1-μm nitrocellulose membrane (GE Healthcare) using a semidry apparatus (ADVANTEC) at 2.5 mA/cm² for 30 min. Chemically synthesized ox and rCrp1 prepared as described (44 (link)) were used as standards. The membranes were blocked by BSA-free StabilGuard Immunoassay Stabilizer (Surmodics) at room temperature for 1 h, incubated with biotinylated anti-Crp1 antibody (1 μg/ml, 76-R29, self-produced), which reacts to both ox and rCrp1–4 and 6 (Fig S2) at 4°C overnight. Membranes were incubated with HRP-conjugated streptavidin (GE Healthcare) at room temperature for 1 h. After incubation, the blots were visualized by Chemi-Lumi One Ultra.

Shimizu Y., Nakamura K., Yoshii A., Yokoi Y., Kikuchi M., Shinozaki R., Nakamura S., Ohira S., Sugimoto R, & Ayabe T. (2020). Paneth cell α-defensin misfolding correlates with dysbiosis and ileitis in Crohn’s disease model mice. Life Science Alliance, 3(6), e201900592.

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AIPL1 Protein Expression Validation

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AIPL1co and AIPLwt protein expression was confirmed with Western blot. Briefly, HEK-293T cells were transfected with pAAV-AIPL1co-His and pAAV-AIPL1wt-His plasmids and Lipofectamine 3000 (ThermoFisher Scientific, Waltham, MA, USA). After 24 h post transfection, cells were harvested and washed in cold PBS and lysed in radioimmunoprecipitation assay (RIPA) buffer. Protein lysate was used for SDS-PAGE using 12% polyacrylamide gel. Then, proteins were transferred onto 0.1 μm nitrocellulose membrane (GE, New York, NY, USA). Free binding sites were blocked with 5% dry milk in PBS-T and treated with goat anti-His antibodies (Abcam, Waltham, MA, USA) for 1 h at room temperature. Anti-tubulin antibodies (Abcam, USA) were used for normalization. Then, secondary anti-rabbit HRP-conjugated antibodies (Abcam, USA) and chemiluminescent substrate Clarity ECL (BioRad, Hercules, CA, USA) were used to visualize proteins with the Chemidoc MP imaging system (BioRad, USA).

Galieva A., Egorov A., Malogolovkin A., Brovin A, & Karabelsky A. (2023). RNA-Seq Analysis of Trans-Differentiated ARPE-19 Cells Transduced by AAV9-AIPL1 Vectors. International Journal of Molecular Sciences, 25(1), 197.

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Quantifying Ubiquitin Dynamics in Rat Lungs

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Rat lungs were homogenized in Radioimmunoprecipitation assay buffer (RIPA lysis buffer) (Abcam; Cambridge, MA) supplemented with a protease inhibitor cocktail (Roche; Indianapolis, IN). Following centrifugation at 12,000 rpm for 20 min at 4 °C, soluble supernatant fractions were collected for total protein and Western blot analysis. Total protein concentrations were determined by BCA assay kit (Thermo Scientific, Waltham, MA). 10 μg total protein were resolved in pre-casted 4–15% gradient Tris-Glycine ‘Stain-Free’ Mini-Protean gel (Bio-Rad, Hercules, CA) to assess for lysine (K)48-linked ubiquitin (1:1000, R&D Systems) and ubiquitin-C (1:1000, Invitrogen) expression. Gels were transferred to 0.1 μm nitrocellulose membrane (GE Healthcare). Horseradish peroxidase (HRP) and SuperSignal West Pico chemiluminescent substrates (Thermo Scientific) were used to detect protein signal intensity. Semi-quantification was performed using Image Lab software (Bio-Rad, Hercules, CA) and normalized relative to total protein imaged from the ‘Stain-Free’ gel.

Wang J., Zhang T., Johnston C.J., Kim S.Y., Gaffrey M.J., Chalupa D., Feng G., Qian W.J., McGraw M.D, & Ansong C. (2020). Protein thiol oxidation in the rat lung following e-cigarette exposure. Redox Biology, 37, 101758.

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MPTP-Induced α-Synuclein and TREM2 Analysis

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For western blotting of α-synuclein, mice were killed 24 h after the last MPTP-subacute administration. The ventral midbrain (VM) was excised and homogenized in RIPA buffer (Thermo Fisher) supplemented with protease inhibitor (Thermo Fisher) using a TissueRuptor (Qiagen, Germantown, MD). Protein concentration was determined using a BCA Protein Assay Kit (Thermo Fisher) according to the manufacturer’s instructions. Samples were diluted to 2 µg/µl in the same extraction buffer. For TREM2, mice were killed at day 4 after MPTP-acute administration. The striatum (St) was excised, and proteins were extracted and quantified as previously described.
30 μg of protein per sample were resolved in a Mini-PROTEAN TGX 4–20% gradient gel and transferred to a 0.1 μm nitrocellulose membrane (GE Healthcare, Chicago, IL, USA). Membranes were incubated overnight with primary antibody for mouse a-synuclein (1:1000, Cell Signaling) or TREM2 (1:1000, Cell Signaling). 4–5 mice per group were used.

Amo-Aparicio J., Daly J., Højen J.F, & Dinarello C.A. (2023). Pharmacologic inhibition of NLRP3 reduces the levels of α-synuclein and protects dopaminergic neurons in a model of Parkinson’s disease. Journal of Neuroinflammation, 20, 147.

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Crps Protein Nitrocellulose Membrane Blot

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50 ng of chemically synthesized Crps were dissolved in 5% acetic acid and pipetted onto a 0.1-μm nitrocellulose membrane (GE Healthcare). The membrane was dried and treated as described for AU-PAGE Western blot.

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Protein Extraction and Quantification Protocol

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Samples for the protein analyses were harvested and extracted in a buffer containing 0.1% NP-40, 0.25 M NaCl, 5 mM EDTA, 50 mM HEPES of 7.5 pH, 0.1%, 0.5 mM DTT, and protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Protein concentrations were assessed by spectrophotometry at 280 nm (DeNovix, Wilmington, DE), and 40 μg of protein extract was loaded onto an SDS-PAGE gel, electrophoresed, transferred to a 0.1-μm nitrocellulose membrane (GE Healthcare, Chicago, IL), and immunoblotted with anti-FXN mouse antibody 17A11 (Abcam, Cambridge, UK) or anti-GAPDH mouse 6C5 antibody (Merck, Darmstadt, Germany) followed by immunoblotting with peroxidase conjugated donkey anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). Quantitation of immunoblots was performed using ImageJ software (National Institutes of Health, Bethesda, MD).

Misiorek J.O., Schreiber A.M., Urbanek-Trzeciak M.O., Jazurek-Ciesiołka M., Hauser L.A., Lynch D.R., Napierala J.S, & Napierala M. (2020). A Comprehensive Transcriptome Analysis Identifies FXN and BDNF as Novel Targets of miRNAs in Friedreich’s Ataxia Patients. Molecular Neurobiology, 57(6), 2639-2653.

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