0.2 mm nitrocellulose membrane

Manufactured by Bio-Rad

Sourced in United States

0.2-mm nitrocellulose membranes are a type of laboratory equipment used for protein transfer and immobilization. These membranes have a pore size of 0.2 millimeters and are made of nitrocellulose material.

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Lab products found in correlation

Ripa buffer, by Thermo Fisher Scientific (2 mentions) Chemidoc mp imaging system, by Bio-Rad (2 mentions) Criterion gel system, by Bio-Rad (1 mentions) Ecl kit, by Cytiva (1 mentions) Ecl substrate solution, by Thermo Fisher Scientific (1 mentions) Blocking buffer, by Bio-Rad (1 mentions) Mouse anti β actin, by Cell Signaling Technology (1 mentions) Mouse anti α tubulin, by Cell Signaling Technology (1 mentions) Chemiluminescence substrate, by Bio-Rad (1 mentions) Mouse anti phospho p38, by Cell Signaling Technology (1 mentions) Rabbit anti phospho nf kb, by Cell Signaling Technology (1 mentions) Imager, by Uvitec (1 mentions) Precast polyacrylamide gel, by Bio-Rad (1 mentions) Rabbit anti gapdh, by Cell Signaling Technology (1 mentions) Protease and phosphatase inhibitor cocktail, by Roche (1 mentions) Tris glycine gel, by Bio-Rad (1 mentions) Sds sample buffer, by Bio-Rad (1 mentions) Irdye 680rd goat anti rabbit igg, by LI COR (1 mentions) Blocking buffer, by LI COR (1 mentions) Irdye 800cw goat anti mouse igg, by LI COR (1 mentions)

5 protocols using 0.2 mm nitrocellulose membrane

Western Blot Analysis of RahU Protein

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Equal volumes of 25 μl of protein extracts in Laemmli buffer were separated on 8–16% sodium dodecyl sulphate-polyacrylamide gel electrophoresis gels using the Criterion gel system (Bio-Rad). Proteins were transferred by electroblotting onto 0.2-mm nitrocellulose membranes (Bio-Rad) at 100 V for 45 min. Membranes were blocked with 5% (w/v) fat-free milk in PBS-T [10-mM phosphate buffered saline (PBS) (pH 7.4) with 0.05% Tween-20] for 1 h at room temperature after which blots were probed with anti-recombinant-RahU (PA0122) mouse serum (52 (link)) diluted 1:2000 in PBS-T, and incubated overnight at 4°C. Immunodetection was performed with peroxidase-conjugated rabbit anti-mouse immunoglobulin G secondary antibody (Sigma) at a dilution of 1:5000 in PBS-T. The blots were then washed three times with PBS-T followed by PBS for 5 min each. Finally, the peroxidase reaction product was visualized using enhanced chemiluminescence (ECL Kit) according to the manufacturer's protocol (Amersham).

Kulkarni P.R., Jia T., Kuehne S.A., Kerkering T.M., Morris E.R., Searle M.S., Heeb S., Rao J, & Kulkarni R.V. (2014). A sequence-based approach for prediction of CsrA/RsmA targets in bacteria with experimental validation in Pseudomonas aeruginosa. Nucleic Acids Research, 42(11), 6811-6825.

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Immunoblotting of Protein Signaling Cascades

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Spinal cords were homogenized in RIPA lysis buffer supplemented with complete proteinase inhibitor cocktail and PhoSTOP phosphatase inhibitors. Primary cells were lysed in RIPA buffer. Protein lysates were cleared of insoluble material through centrifugation, and the resulting protein lysates were subjected to SDS-PAGE. Proteins were wet transferred to 0.2 mm nitrocellulose membranes (BioRad Laboratories), which were blocked using 5% non-fat milk in 1% TBS-T buffer for 1 hr at room temperature. The membranes were incubated overnight using the following primary antibodies: anti-actin HRP, anti-Histone H3, anti-p65, anti-pp65, anti-STAT1, anti-pSTAT1, anti-ZAP70, anti-pZAP70, anti-PLC-g1, anti-pPLC-g1, anti-NFAT1, anti-mTOR, anti-pmTOR, anti-AKT, anti-pAkt, anti-Akt, anti-4EBP1, anti-p4EBP1, anti-S6K, anti-pS6K, anti-c-Myc, anti-pc-Myc, anti-JNK, anti-pJNK, anti-ERK½ or anti-pERK½. All primary antibodies were used at a 1:1,000 dilution in 5% non-fat milk. Membranes were washed in TBS-T and incubated with the following appropriate secondary antibodies: goat anti-rabbit-HRP or goat anti-mouse HRP. All secondary antibodies were used at a 1:40,000 dilution in 5% non-fat milk. Protein bands were visualized following exposure of the membranes to ECL substrate solution (ThermoFisher) and quantified by densitometryc analysis using ImageJ software.

Uchimura T., Oyama Y., Deng M., Guo H., Wilson J.E., Rampanelli E., Cook K.D., Misumi I., Tan X., Chen L., Johnson B., Tam J., Chou W.C., Brickey W.J., Petrucelli A., Whitmire J.K, & Ting J.P. (2018). The Innate Immune Sensor NLRC3 Acts as a Rheostat that Fine-Tunes T Cell Responses in Infection and Autoimmunity. Immunity, 49(6), 1049-1061.e6.

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Protein Isolation and Western Blotting

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For protein isolation, TSCs were lysed using RIPA buffer (Thermo Fisher Scientific, 89, 900) . For western blotting, proteins were transferred to 0.2mm Nitrocellulose membranes (Bio-Rad, 1,620,112), and membranes were then blocked with 0.1% PBS-T (PBS plus 0.1% Triton X-100) containing 5% skim milk at RT for 1 h. To detect specific proteins of interest, WNT7B and a-TUBULIN antibodies were used (See Methods S4). Horseradish peroxidase (HRP)-conjugated secondary antibodies were then used. Each antibody incubation was followed by washing with 0.1% PBS-T. Luminescence was detected with a Bio-Rad ChemiDoc MP Imaging System.

Seong J., Frias-Aldeguer J., Holzmann V., Kagawa H., Sestini G., Heidari Khoei H., Scholte Op Reimer Y., Kip M., Pradhan S.J., Verwegen L., Vivié J., Li L., Alemany A., Korving J., Darmis F., van Oudenaarden A., Ten Berge D., Geijsen N, & Rivron N.C. (2022). Epiblast inducers capture mouse trophectoderm stem cells in vitro and pattern blastoids for implantation in utero. Cell stem cell, 29(7).

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Evaluating Cellular Signaling Pathways

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The HUVECs were lysed with the RIPA solution (0.1% SDS, 150 mM NaCl, 1.0% Triton X-100, 10 mM Tris, 5 mM EDTA pH 8.0), to which we added a protease and phosphatase inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA). In each sample, the protein concentration was evaluated using a Bradford assay. Proteins (25 µg) were separated by 4–15% precast polyacrylamide gel (Bio-Rad, Hercules, CA, USA), and then transferred to a 0.2 mm nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). A blocking buffer (Bio-Rad, Hercules, CA, USA) was used to block the membrane, which was then incubated overnight with primary antibodies. Mouse anti-phospho-p38 (Cell Signaling), rabbit anti-phospho-NF-kB (Cell Signaling), mouse anti-ICAM-1 (Cell Signaling), mouse anti-β-actin, mouse anti-α-tubulin, and rabbit-anti-GAPDH (Cell Signaling) were used as the primary antibodies. Anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibodies were used as the secondary antibodies (The Jackson laboratory, Bar Harbor, ME, USA). A Uvitec Imager (UVItec, Cambridge, UK) was used to distinguish the protein bands, using a chemiluminescence substrate (Bio-Rad), that were then quantified using ImageJ software. Each measure was normalized versus β-actin, α-tubulin, or GAPDH.

Matacchione G., Borgonetti V., Ramini D., Silvestrini A., Ojetti M., Galeotti N, & Olivieri F. (2023). Zingiber officinale Roscoe Rhizome Extract Exerts Senomorphic and Anti-Inflammatory Activities on Human Endothelial Cells. Biology, 12(3), 438.

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Western Blot Analysis of Protein Lysates

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Protein lysates were prepared from the livers of mice by homogenization in SDS sample buffer (Biorad, Hercules, CA) containing β-mercaptoethanol (Sigma) or cell scraping in AML12 cells. Approximately 30 μg of total protein was resolved on a 4–20% Tris-glycine gel (Biorad) and transferred onto a 0.2 mM nitrocellulose membrane (Biorad). Membranes were blocked with blocking buffer (LI-COR Biosciences, Lincoln, NE) and incubated overnight with primary antibodies as indicated. Secondary antibodies IRDye 800CW Goat anti-Mouse IgG (LI-COR, 926–32210) and IRDye 680RD Goat anti-Rabbit IgG (LI-COR, 926–68071) were used to detect proteins of interest via the ChemiDoc MP Imaging System (Biorad).

Mainali R., Buechler N., Otero C., Edwards L., Key C.C., Furdui C, & Quinn M.A. (2024). Itaconate stabilizes CPT1a to enhance lipid utilization during inflammation. eLife, 12, RP92420.

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