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Trans blot turbo midi nitrocellulose membrane

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The Trans Blot Turbo Midi Nitrocellulose membranes are designed for the transfer of proteins from polyacrylamide gels to a nitrocellulose membrane. The membranes are compatible with the Trans-Blot Turbo Transfer System and facilitate the efficient and rapid transfer of proteins from the gel to the membrane.

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Lab products found in correlation

Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Mini protean tgx stain free precast gel, by Bio-Rad (1 mentions) Ab76495, by Abcam (1 mentions) Sc 2005, by Santa Cruz Biotechnology (1 mentions) Amersham hyperfilm ecl, by GE Healthcare (1 mentions) Amersham ecl western blotting detection reagent, by GE Healthcare (1 mentions) Sc 2004, by Santa Cruz Biotechnology (1 mentions) Ab6276, by Abcam (1 mentions) Mini protean tgx precast gel, by Bio-Rad (1 mentions) Westernbright sirius hrp substrate, by Advansta (1 mentions) Anti rabbit igg hrp linked 7074s, by Cell Signaling Technology (1 mentions) Chemidoc gel imaging system, by Bio-Rad (1 mentions) Myc tag 9b11, by Cell Signaling Technology (1 mentions) Criterion tgx gel, by Bio-Rad (1 mentions) Protease and phosphatase inhibitor, by Merck Group (1 mentions) Bca assay, by Thermo Fisher Scientific (1 mentions) Nupage 4 to 12 bis tris gel, by Thermo Fisher Scientific (1 mentions) Ripa buffer, by Boston BioProducts (1 mentions) Ripa lysis buffer, by Boston BioProducts (1 mentions) Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)

6 protocols using trans blot turbo midi nitrocellulose membrane

1

Protein extraction and immunoblotting analysis

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Total protein extraction and quantification was carried out as previously described8 (link). Forty µg of heat-denatured proteins were separated by electrophoresis using 10% Mini-PROTEAN® TGX Stain-Free™ Precast Gels (Bio-Rad Laboratories Ltd., Hempstead, UK). Separated proteins were transferred onto a Trans-Blot® Turbo™ Midi Nitrocellulose membrane (Bio-Rad Laboratories Ltd.), and incubated with 1:1000 primary antibodies (anti-ETV5, clone 3B10, Novus Biologicals Ltd, Cambridge, UK; anti-FGFR3, sc-13121, Insight Biotechnology Ltd, Middlesex; anti-phospho-ERK1/2 (Tyr 204), sc-7383, Insight Biotechnology Ltd, Middlesex; anti-YAP/TAZ, D24E4, Cell Signalling Technology Inc.; anti-Laminin B1, Ab16048, Abcam, Cambridge, UK; anti-beta-actin, sc-81178, Insight Biotechnology Ltd; anti-tubulin alpha, MorphoSys UK Ltd., Kidlington, UK) overnight at 4 °C. Bound primary antibody was detected using HRP-conjugated secondary antibody and Luminata Forte Western HRP Substrate (Millipore UK Limited, Watford, UK). Extraction of proteins from specific subcellular fractionation was performed using the Subcellular Protein Fractionation Kit for Cultured Cells (Fisher Scientific Ltd., Loughborough).
di Martino E., Alder O., Hurst C.D, & Knowles M.A. (2019). ETV5 links the FGFR3 and Hippo signalling pathways in bladder cancer. Scientific Reports, 9, 5740.
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2

Western Blot Analysis of Tumor Proteins

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Tumor tissue was lysed in radioimmunoprecipitation assay buffer with a Precellys 24 homogenizer/lysing kit (1.4 mm ceramic beads; Bertin Instruments). Polyacrylamide gel electrophoresis was performed with 4%-15% Mini-Protean TGX Precast Gels (BioRad), 15-well. After transfer to a Trans-Blot Turbo Midi Nitrocellulose membrane (Bio-Rad), proteins were probed overnight at 4°C with primary antibodies against TK1 (1:5,000; ab76495 [Abcam]), TS (1:1,000; 3766S [Cell Signaling Technologies]), TP (1:2,000; 12383-1AP [Acris]), or actin (1:10,000; ab6276 [Abcam]). Goat anti-rabbit (1:10,000; sc-2004 [Santa Cruz]) and goat anti-mouse (1:10,000; sc-2005 [Santa Cruz]) were used as secondary antibodies. Signals were then revealed with Amersham ECL Western blotting detection reagents and Amersham Hyperfilm ECL (both GE Healthcare). ImageJ was used for quantification via densitometry, and protein levels are expressed relative to an actin loading control.
Schelhaas S., Wachsmuth L., Hermann S., Rieder N., Heller A., Heinzmann K., Honess D.J., Smith D.M., Fricke I.B., Just N., Doblas S., Sinkus R., Döring C., Schäfers K.P., Griffiths J.R., Faber C., Schneider R., Aboagye E.O, & Jacobs A.H. (2018). Thymidine Metabolism as a Confounding Factor for 3'-Deoxy-3'-18F-Fluorothymidine Uptake After Therapy in a Colorectal Cancer Model. Journal of nuclear medicine : official publication, Society of Nuclear Medicine, 59(7).
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3

Western Blotting of Viral Proteins

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Proteins were separated in 4–15% Criterion TGX gels (Bio-Rad, Hercules, CA, USA) and transferred to Trans Blot Turbo Midi Nitrocellulose membranes (Bio-Rad). The membranes were blocked with 5% non-fat dry milk in TBS-T. Antibodies detecting target proteins were diluted in 5% non-fat dry milk in TBS-T and incubated overnight, at 4 °C. Antibodies detecting ORF57 (sc-135746), ORF45 (sc-53883), K8.1 (sc-65446), Actin (sc-8432) were from Santa Cruz Biotechnology (SCBT, Dallas, TX, USA). Antibodies detecting HA tag (HA.11) were from Biolegend (San Diego, CA, USA), Myc-tag (9B11) from Cell Signaling Technology (Danvers, MA, USA) and ORF50 was a gift from Carolina Arias (UC Santa Barbara, Santa Barbara, CA, USA). Anti-Rabbit IgG HRP-linked (7074S) and anti-Mouse IgG HRP-linked (7076S) secondary antibodies were from Cell Signaling Technology. WesternBright Sirius HRP substrate (Advansta, San Jose, CA, USA) was used for revealing the protein bands during imaging in a ChemiDoc gel imaging system (Bio-Rad).
Elbasani E., Falasco F., Gramolelli S., Nurminen V., Günther T., Weltner J., Balboa D., Grundhoff A., Otonkoski T, & Ojala P.M. (2020). Kaposi’s Sarcoma-Associated Herpesvirus Reactivation by Targeting of a dCas9-Based Transcription Activator to the ORF50 Promoter. Viruses, 12(9), 952.
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4

Protein Extraction and Analysis

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Cells were lysed in RIPA buffer (Boston BioProducts), supplemented with protease and phosphatase inhibitors (Sigma-Aldrich) and sonicated for 1 minute. Protein concentration was determined by bicinchoninic acid (BCA) assay (Thermo Fisher Scientific), samples were subjected to SDS-PAGE by using NuPAGE 4 to 12% Bis-Tris gels (Life Technologies) and blotted on Trans-Blot Turbo Midi Nitrocellulose membranes (Bio-Rad Laboratories). The list of the antibodies with their relative dilutions is reported in the Supplementary Table S8.
Guarducci C., Nardone A., Russo D., Nagy Z., Heraud C., Grinshpun A., Zhang Q., Freelander A., Leventhal M.J., Feit A., Cohen Feit G., Feiglin A., Liu W., Hermida-Prado F., Kesten N., Ma W., De Angelis C., Morlando A., O'Donnell M., Naumenko S., Huang S., Nguyen Q.D., Huang Y., Malorni L., Bergholz J.S., Zhao J.J., Fraenkel E., Lim E., Schiff R., Shapiro G.I, & Jeselsohn R. (2024). Selective CDK7 Inhibition Suppresses Cell Cycle Progression and MYC Signaling While Enhancing Apoptosis in Therapy-resistant Estrogen Receptor–positive Breast Cancer. Clinical Cancer Research, 30(9), 1889-1905.
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5

Western Blot Quantification of Protein Levels

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Cell pellets were lysed with RIPA lysis buffer (Boston BioProducts) freshly supplemented with protease inhibitor cocktail (Sigma-Aldrich). Protein lysate was standardized using the BCA protein quantitation assay kit as per manufacturer's instructions (ThermoFisher Scientific, Waltham, MA, USA), and 20-40 μg whole protein lysates were resolved on either 10% or 4-12% NuPage Bis-Tris gels (Life Technologies) and blotted on Trans-Blot® Turbo™ Midi Nitrocellulose membranes (Bio-Rad Laboratories). Membranes were blocked with 5% (wt/vol) BSA in Tris-buffered saline with Tween-20 (20 mM Tris-HCl (pH 7.6), 137 mM NaCl, 0.1% Tween-20), then incubated overnight at 4 °C with the following primary antibodies: p53 (1:1000, DO-1; Sigma-Aldrich); p21 (1:1000, 12D1; Cell Signaling Technologies) and GAPDH (D16H11; Cell Signaling Technologies). Appropriate horseradish peroxidase-conjugated secondary antibodies (1:2500; Santa Cruz Biotechnologies) were diluted in Tris-buffered saline with 0.1% Tween-20 and membranes were probed at room temperature for 2 h. Immunoblots were visualized with Pico ECL Chemiluminescence reagents (ThermoFisher Scientific). Figures of the gel and molecular weight markers are in Supplementary Fig. 5.
Grinshpun A., Russo D., Ma W., Verma A., Hermida-Prado F., Sherman S., Gaglia G., Kabraji S., Kirkner G., Hughes M.E., Lin N.U., Sandusky Z., Nardone A., Guarducci C., Nguyen Q.D., Santagata S., Nagy Z, & Jeselsohn R. (2024). Pure estrogen receptor antagonists potentiate capecitabine activity in ESR1-mutant breast cancer. NPJ breast cancer, 10(1).
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6

Quantitative Analysis of Mutant SOD1 Aggregation

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Western blots were performed according to standard procedures in Criterion™ TGX™ precast gels (BioRad Laboratories) and Trans‐Blot® Turbo™ Midi Nitrocellulose membranes (BioRad Laboratories). Primary rabbit anti‐human SOD1 antibodies (Jonsson et al., 2006 (link)) were used for the detection of total mutant SOD1 content in the cell extracts and detected with secondary horseradish peroxidase‐conjugated polyclonal anti‐rabbit IgG (Dako). Reactivity was detected using ECL chemiluminescent detection reagent (GE Healthcare) and recorded using ChemiDoc imager (BioRad Laboratories). Quantification of band intensities was performed using ImageLab software (BioRad Laboratories).
The total protein content in the cell extracts used for BEM analysis was determined with Pierce™ BCA Protein Assay Kit (Thermo Fisher scientific) and used to normalize the amounts of aggregation to relative protein content.
Nordström U., Lang L., Ekhtiari Bidhendi E., Zetterström P., Oliveberg M., Danielsson J., Andersen P.M, & Marklund S.L. (2022). Mutant SOD1 aggregates formed in vitro and in cultured cells are polymorphic and differ from those arising in the CNS. Journal of Neurochemistry, 164(1), 77-93.
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