Placental proteins were extracted as described above. After removal of the organic (lipid) phase, the protein was pelleted in methanol and dissolved in RIPA buffer overnight. After sonication of lysate, protein concentration was determined using a Pierce BCA protein assay kit (Thermo Fisher Scientific Inc. Waltham, MA). Proteins were resolved in 4–15% SDS-PAGE and transferred to polyvinylidene difluoride membranes using Trans-Blot Turbo RTA Midi PVDF Transfer Kit (Bio-Rad). Membranes were blocked in 5% BSA/TBS-T followed by overnight incubation in primary antibodies against GLUT1, LPL, CD36, FABP3, and ADRP followed by one-hour incubation in HRP-conjugated goat anti-rabbit IgG secondary antibody (SouthernBiotech, Birmingham, AL). Luminata Forte Western HRP substrate (EMD Millipore, Billerica, MA) and VisionWorks LS software (UVP, Upland, CA) were used for imaging and recording integrated optical densities. Readings were normalized to β-actin. Antibody information is shown in Supplemental Table S2 .
Trans blot turbo rta midi pvdf transfer kit
Manufactured by Bio-Rad
Sourced in United States
The Trans-Blot Turbo RTA Midi PVDF Transfer Kit is a laboratory equipment designed for the rapid and efficient transfer of proteins from polyacrylamide gels to polyvinylidene fluoride (PVDF) membranes. The kit includes all the necessary components for the transfer process, including the transfer pack and buffer solutions.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc mp imaging system, by Bio-Rad (3 mentions)
Clarity western ecl substrate, by Bio-Rad (3 mentions)
Hrp conjugated goat anti rabbit igg secondary antibody, by Southern Biotech (2 mentions)
Luminata forte western hrp substrate, by Merck Group (2 mentions)
Visionworks ls software, by Analytik Jena (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Tgx stain free fast cast acrylamide kit 7, by Bio-Rad (1 mentions)
Bradford reagent, by Bio-Rad (1 mentions)
Tbs t, by Merck Group (1 mentions)
Tgx stain free fast cast acrylamide kit 10, by Bio-Rad (1 mentions)
Criterion tgx stain free precast gel, by Bio-Rad (1 mentions)
Tris glycine sds running buffer, by Bio-Rad (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Chemidoc mp system, by Bio-Rad (1 mentions)
Trans blot turbo, by Bio-Rad (1 mentions)
6 protocols using trans blot turbo rta midi pvdf transfer kit
1
Placental Protein Extraction and Analysis
2
Protein Extraction and Western Blot Analysis
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At the end of the culture, cells in a monolayer or seeded in 3D scaffolds were washed with phosphate-buffered saline (PBS), and then protein extraction was performed using radioimmunoprecipitation (RIPA) lysis buffer supplemented with several protease inhibitors as previously described [45 (link)]. Bradford reagent was used to determine the protein concentration (Bio-Rad; Hercules, CA, USA). The electrophoreses were performed (10 μg of the proteins extracts) using 7.5% polyacrylamide gels (TGX Stain-Free Fast Cast Acrylamide Kit 7.5%; Bio-Rad; Hercules, CA, USA). Then, proteins were transferred to a Polyvinylidenedifluoride membrane (Trans-Blot Turbo RTA Midi PVDF Transfer Kit, Bio-Rad). We used 10% non-fat milk diluted in Tris-buffered saline with 0.1% Tween (TBST, Sigma Aldrich; Saint Louis, MO, USA) to block unspecific binding sites of the membranes. Then, membranes were incubated overnight at 4 °C with primary antibodies and then for 1 h at room temperature with secondary antibodies. All antibodies are listed in Table 2 . The chemiluminescence signal (Clarity Western ECL Substrate, Bio-Rad; Hercules, CA, USA) was detected on the ChemiDoc MP Imaging System (Bio-Rad; Hercules, CA, USA).
3
Western Blot Protein Expression Analysis
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Protein extracts (12 µg per sample) were resolved on 10% polyacrylamide gels (TGX Stain Free Fast Cast Acrylamide Kit 10%, Bio-Rad, Hercules, CA, USA) and transferred to a polyvinylidene difluoride membrane (Trans-Blot Turbo RTA Midi PVDF Transfer Kit, Bio-Rad, Hercules, CA, USA). Membranes were incubated with 10% non-fat milk powder in Tris-buffered saline with 0.1% Tween (TBST) for 1 h to block unspecific binding sites. Then, membranes were incubated overnight at 4 °C under agitation with antibodies listed in Supplementary Table S2 . The next day, membranes were washed with TBST and incubated for 1 h with the corresponding secondary HRP-conjugated goat anti-rabbit or anti-mouse IgG antibody (Jackson Immunoresearch). Proteins were visualized with an enhanced Clarity Western ECL Substrate (Bio-Rad, Hercules, CA, USA) using the ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA). Protein expression was measured by quantifying the density of immunoblots bands calculated relative to GAPDH using ImageJ® (NIH Image, Bethesda, MD, USA), and expressed as arbitrary units (AU).
4
Western Blot Analysis of Tumor Proteins
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Standard Radioimmunoprecipitation assay (RIPA) buffer was used to harvest protein lysates from tumour tissues while a Bradford assay was performed to determine protein concentration. Protein samples (50 μg), prepared with Laemmli’s sample buffer, were loaded onto 4–20% Criterion™ TGX Stain-Free™ Precast Gels (BioRad, CA, USA). Proteins were separated at 100 V for 10 min and 120 V for 60 min in Tris/Glycine/SDS running buffer (BioRad, CA, USA). Proteins were transferred onto Polyvinylidene difluoride (PVDF) membranes (Trans-Blot® Turbo RTA Midi PVDF transfer kit, BioRad, CA, USA) with the Trans-Blot® Turbo Transfer System (BioRad, CA, USA) with the conditions of 25 V, 1.0A, 30 min. The Stain-Free™ properties of the membranes were utilized on the Chemidoc™ MP System (BioRad, CA, USA) to determine the total protein intensities of each membrane. Membranes were blocked in 5% milk for 1 h and incubated in primary antibody, prepared in tris-buffered saline with tween 20 (TBS-T), at 4 °C overnight. On the following day membranes were incubated in secondary antibody, prepared in TBS-T, for 1 h at RT. Antibody details are listed in the Additional file 1 . After incubation, membranes were developed on the Chemidoc™ MP System with Clarity™ Electrochemiluminescence (ECL) Substrate (BioRad, CA, USA).
5
Placental Protein Extraction and Analysis
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Placental proteins were extracted as described above. After removal of the organic (lipid) phase, the protein was pelleted in methanol and dissolved in RIPA buffer overnight. After sonication of lysate, protein concentration was determined using a Pierce BCA protein assay kit (Thermo Fisher Scientific Inc. Waltham, MA). Proteins were resolved in 4–15% SDS-PAGE and transferred to polyvinylidene difluoride membranes using Trans-Blot Turbo RTA Midi PVDF Transfer Kit (Bio-Rad). Membranes were blocked in 5% BSA/TBS-T followed by overnight incubation in primary antibodies against GLUT1, LPL, CD36, FABP3, and ADRP followed by one-hour incubation in HRP-conjugated goat anti-rabbit IgG secondary antibody (SouthernBiotech, Birmingham, AL). Luminata Forte Western HRP substrate (EMD Millipore, Billerica, MA) and VisionWorks LS software (UVP, Upland, CA) were used for imaging and recording integrated optical densities. Readings were normalized to β-actin. Antibody information is shown in Supplemental Table S2 .
6
Western Blot Analysis of Extracellular Vesicles and Extracts
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Protein extracts (from eACs and exosomes) were mixed with loading buffer, denatured at 95 °C for 5 min and separated via electrophoresis on 7.5% (eAC protein extracts) and 12% (EV characterization) polyacrylamide gels (TGX Stain-Free Fast Cast Acrylamide Kit 7.5% and 12%; Bio-Rad). Proteins were transferred onto a PVDF membrane (Trans-Blot Turbo RTA Midi PVDF Transfer Kit; Bio-Rad) using a transfer system (Trans-blot® Turbo™; Bio-Rad). Unspecific site blockage of PVDF membranes was carried out at RT with 10% non-fat milk diluted in TBST and membranes were incubated overnight at 4 °C with primary antibodies. After three washes of 5 min in TBST, membranes were incubated for 1 h at RT with secondary antibodies and then rinsed again with TBST. All antibodies and corresponding dilutions are listed in Table 2 for EV characterization, and in Table 3 for eAC phenotype analysis. The chemiluminescence signal (Clarity Western ECL Substrate; Bio-Rad) was detected with the ChemiDoc MP Imaging System (Bio-Rad).
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