Trans blot membrane

Manufactured by Bio-Rad

Sourced in United States

The Trans-blot membrane is a key component in the protein transfer process, enabling the efficient and reliable transfer of proteins from SDS-PAGE gels to a membrane support for subsequent analysis and detection.

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6 protocols using trans blot membrane

Western Blot Analysis of Mouse Liver Proteins

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Mouse liver nuclear and whole cell extracts containing equal amounts of protein were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to Trans-blot membranes (Bio-Rad, Hercules, CA). Membranes were blocked for 1 h at room temperature in 5% nonfat dry milk or 5% BSA (for phosphoproteins) dissolved in Tris-buffered saline with Tween-20 (TBST) prior to incubation with primary antibodies. Membranes were subsequently washed and probed with a goat anti-rabbit IgG-AP secondary antibody (1:2000) for 1 h at room temperature. The blots were then washed with TBST and incubated with Tropix CDP Star^® Nitro block II™ ECL reagent according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA). Protein bands were analyzed and quantified by FluorChem FC Imaging System (Alpha Innotech, San Leandro, CA).

Mallick P., Basu S., Moorthy B, & Ghose R. (2017). Role of Toll-like receptor 4 in Drug-drug Interaction between Paclitaxel and Irinotecan in vitro. Toxicology in vitro : an international journal published in association with BIBRA, 41, 75-82.

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Western Blotting Protocol for Testicular Proteins

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Testes were homogenated using a Tissue‐Tearor (RPI Corp, Mt. Prospect, IL, USA) in lysis buffer containing 50 mmol/L Tris‐HCl, 150 mmol/L NaCl, 1% NP‐40, 1 mmol/L EDTA, 2 mmol/L MgCl₂, 0.5% sodium deoxycholate, complete protease inhibitor and phosphorylase inhibitor cocktail (Roche, Mannheim, Germany). The protein concentrations were determined using Bradford protein assay kits (Bio‐Rad, Hercules, CA, USA). 20 μg testicular homogenates each sample was electrophoresed in 10% SDS/PAGE gels and then electroblotted to TransBlot membranes (Bio‐Rad). After blocking with 3% non‐fat milk in Tris‐buffered Saline‐Tween‐20, membranes were incubated with the primary antibody as listed in Table 2, followed by horseradish peroxidase (HRP)‐conjugated secondary antibodies (Santa Cruz Biotech). Signal was detected using a Pierce ECL Western blotting substrate detection kit (Thermo Fisher Scientific, Waltham, MA, USA). All membranes were reblotted with β‐actin or glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) antibody as the loading control. The intensity of specific bands was scanned using image analysis software Total Lab. The results were presented as the ratio of target protein over β‐actin or GAPDH.

Guo K., He Y., Liu L., Liang Z., Li X., Cai L., Lan Z., Zhou J., Wang H, & Lei Z. (2018). Ablation of Ggnbp2 impairs meiotic DNA double‐strand break repair during spermatogenesis in mice. Journal of Cellular and Molecular Medicine, 22(10), 4863-4874.

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Western Blot Analysis of Signaling Proteins

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Cultivated cells were harvested and lysed using lysis buffer [1% (v/v) Triton X-100, 200 mM HEPES (pH 7.9), 300 mM NaCl, 100 mM KCl, 10 mM EDTA, 10 µg/ml aprotinin, 100 µg/ml leupeptin, and 10 µM PMSF]. Twenty micrograms of total protein lysate was electrophoresed in an acrylamide gel and transferred to a TransBlot^® membrane (162–0145, Bio-Rad, Hercules, CA, USA). Membranes were blocked in 5% (w/v) milk, incubated with primary antibodies for 1 h at room temperature, washed and incubated for 1 h with the appropriate HRP-conjugated secondary antibodies (sc-2004 and sc2005, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The blots were washed in PBS containing 0.05% (v/v) Tween-20^® (P1379, Sigma-Aldrich Korea) and an ECL kit (34080, Pierce Biotechnology, Rockford, IL, USA) was used prior to detection of protein bands. The antibodies used were: rabbit anti-prohibitin (sc-28259, Santa Cruz Biotechnology, Inc.), rabbit anti-β-catenin (sc-7199, Santa Cruz Biotechnology, Inc.), rabbit anti-GSK-3β (sc-9166, Santa Cruz Biotechnology, Inc.), rabbit anti-p-GSK-3β (sc-11757-R, Santa Cruz Biotechnology, Inc.), mouse anti-c-Myc (sc-40, Santa Cruz Biotechnology, Inc.), and mouse anti-β-actin monoclonal (sc-8432, Santa Cruz Biotechnology, Inc.) to determine equal loading.

Kim D.M., Jang H., Shin M.G., Kim J.H., Shin S.M., Min S.H, & Kim I.C. (2017). β-catenin induces expression of prohibitin gene in acute leukemic cells. Oncology Reports, 37(6), 3201-3208.

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Western Blot Immunodetection of Mitochondrial Proteins

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From SDS-tricine-PAGE, proteins were electrotransferred onto a nitrocellulose Trans-Blot membrane (Bio-Rad) for immunodetection. Membranes were washed, blocked, and independently incubated for 4 h with the following antibodies: anti-Cox2 antibody at a 1:9000 dilution (Invitrogen; Molecular Probes), anti-Oxa1 antibody at a 1:1000 dilution, anti-Hog1 antibody at a 1:2000 dilution (Santa Cruz Biotechnology), and anti-Atp2 antibody at a 1:50,000 dilution. Alkaline phosphatase-conjugated IgGs (1:15,000 for 2 h) were used as secondary antibodies. Insoluble black–purple precipitates were formed upon addition of nitro-blue tetrazolium chloride and 5-bromo-4-chloro-3′-indolyl phosphate p-toluidine salt. Images of the immunodecorated polypeptide bands were captured in an HP Scanjet G4050. For immunodetection on previously probed membranes using a different primary antibody, membranes were stripped by incubation for 45 min at 50°C in the presence of 2% SDS, 62.5 mM Tris-HCl, pH 6.8, and 100 mM β-mercaptoethanol.

Rubalcava-Gracia D., García-Rincón J., Pérez-Montfort R., Hamel P.P, & González-Halphen D. (2019). Key within-membrane residues and precursor dosage impact the allotopic expression of yeast subunit II of cytochrome c oxidase. Molecular Biology of the Cell, 30(18), 2358-2366.

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Western Blotting of Osteoblast Markers

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Western blot was performed with whole cell lysates from MD10-F2 cells. Cells were washed with cold PBS and lysed with RIPA buffer (1× PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mg/ml phenylmethylsulfonyl fluoride, 30 μl/ml aprotinin, 100 mM sodium orthovanadate). Proteins (40 μg/well) were resolved by 10% SDS-PAGE and transferred to a Trans-blot membrane (Bio-Rad). Western blot was performed as described earlier^{2 (link)}. Anti-Dlx3, anti-Osx, and anti-Dsp (Santa Cruz) was used as primary antibodies. β-actin antibody (Santa Cruz) was used as a loading control.

Yang G., Yuan G., MacDougall M., Zhi C, & Chen S. (2017). BMP-2 induced Dspp transcription is mediated by Dlx3/Osx signaling pathway in odontoblasts. Scientific Reports, 7, 10775.

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MALDI-TOF Analysis of E. coli LPS

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Samples of deep rough (Rd2) LPS from E. coli strain F583 (Sigma, #L6893) were prepared and analyzed by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometery as described in ref 20 (link). A small amount of the LPS was solubilized in a mixture of methanol/water (1:1) containing 5 mM EDTA and dissolved by brief ultrasonication. A few microliters of the obtained mixture were then desalted on a small piece of Parafilm with some grains of cation-exchange beads (BT AG 50W-X8, Bio-Rad), previously converted into the ammonium form. A total of 0.3 μL of this sample solution was deposited, together with the same volume of 20 mM dibasic ammonium citrate, in a thin layer of homogeneous matrix film obtained from a solution, the components of which were 2,4,6-trihydroxyacetophenone (THAP), 200 mg/mL in methanol, and 15 mg/mL nitro-cellulose (Trans-blot membrane, BioRad) in acetone/propan-2-ol (1:1 v/v), mixed in a 4:1 v/v ratio.

Kucharska I., Liang B., Ursini N, & Tamm L.K. (2016). Molecular Interactions of Lipopolysaccharide with an Outer Membrane Protein from Pseudomonas aeruginosa Probed by Solution NMR. Biochemistry, 55(36), 5061-5072.

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