Pieces of AI and PI of roughly 3 cm length were homogenized on ice in lysis buffer (20 mM Tris-HCl, pH 7.5, 10 mM NaF, 150 mM NaCl, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, leupeptin, and trypsin inhibitor 10 μg/mL). Cytosolic protein concentration was spectrophotometrically evaluated by using bovine serum albumin as standard, and 50 μg of proteins were subjected to SDS-PAGE. The blot was performed by transferring proteins from a slab gel to a nitrocellulose membrane using a Bio-Rad Transblot Turbo (Bio-Rad Laboratories, Hercules, CA, USA). The filter was then blocked at room temperature with milk buffer (1X PBS with pH 7.4, 5% non-fat dried milk and 1% NaF) for 45 min and probed at 4 °C overnight with anti-nitrotyrosine (Nox-Tyr, 1:1000, Merck Millipore, Billerica, MA, USA). Western Blot for anti-GAPDH (1:8000, Sigma-Aldrich, Milan, Italy) was performed to ensure equal sample loading. Bands were detected by ChemiDoc Imaging System (Bio-Rad Laboratories, Hercules, CA, USA).
Biorad trans blot turbo
Manufactured by Bio-Rad
Sourced in United States, Germany
The BioRad Trans-Blot-Turbo is a rapid protein transfer system. It is designed to transfer proteins from polyacrylamide gels to membrane supports for further analysis or detection.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated igg secondary antibody, by Santa Cruz Biotechnology (2 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (2 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Cl xposure x ray film, by Thermo Fisher Scientific (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
P c raf, by Cell Signaling Technology (1 mentions)
Rigosertib, by Selleck Chemicals (1 mentions)
Tgx gel, by Bio-Rad (1 mentions)
Chemiluminescence reagent, by PerkinElmer (1 mentions)
Direct detect spectrometer, by Merck Group (1 mentions)
Fusion sl, by Avantor (1 mentions)
Facsaria 2, by BD (1 mentions)
Mini protean tgx sds page gels, by Bio-Rad (1 mentions)
Methanol activated pvdf membrane, by Bio-Rad (1 mentions)
6 protocols using biorad trans blot turbo
1
Quantifying Nitrotyrosine Levels in Cells
2
Western Blot Analysis of Signaling Proteins
3
Western Blot for C7 Protein Quantification
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The cell lysate was prepared as follows: 8 × 105 fibroblasts or keratinocytes were plated in a 100 mm dish to achieve 70–80% confluence the following day. At 48 h after infection, cells were lysed with radioimmunoprecipitation assay buffer42 (link). Lysate was centrifuged at 13,500 ×g for 5 min at 4 °C, and the supernatant was mixed with a 6x Laemmli loading buffer. Before loading onto the SDS–PAGE gel, the samples were heated for 5 min at 95 °C. For C7 detection, 5–30 μg protein was loaded onto a 6% acrylamide gel. The primary antibodies used were C7 polyclonal rabbit antibody (Sigma prestige Ab, cat. no. HPA042420), GAPDH and β-actin (Santa Cruz Biotechnology). Resolved proteins were transferred onto nitrocellulose membrane with a BioRad Trans-Blot-Turbo (BioRad), blocked in PBS and 0.1% Tween with 5% milk or 5% BSA according to the requirements of the primary antibody, and incubated overnight with the primary antibody. After incubation with IgG-HRP-conjugated secondary antibody (Santa Cruz Biotechnology), the membrane was incubated with Pierce ECL western blotting substrate (ThermoFisher Scientific) and exposed to CL-XPosure X-ray film (ThermoFisher Scientific). C7 was quantified by densitometry (ImageJ v1.52), using a known concentration of purified recombinant C7 (donated by Krystal Biotech) for comparison.
4
Rigosertib Signaling Pathway Analysis
5
Retinal Müller Glia Activation Assessment
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To analyze retinal Müller glia activation, for western blots the isolated retinae were homogenized in 90 μl 0.1% SDS lysis buffer and protein concentration was determined by Direct Detect Spectrometer (Merck KGaA, Darmstadt, Germany). Samples were separated in a 4–20% TGX Gel (Bio-Rad Laboratories, München, Germany) and immunoblotted with Bio-Rad Trans-Blot Turbo to a polyvinylidene difluoride membrane (Bio-Rad Laboratories, München, Germany). Non-specific binding was blocked by incubation with 5% non-fat dry milk in TBS, containing 0.1% Tween (Sigma-Aldrich, Darmstadt, Germany), followed by overnight incubation at 4 °C with rabbit anti-rat glial fibrillary acidic protein (GFAP) (1:400, Santa Cruz, Heidelberg, Germany) or rabbit anti-rat beta Tubulin (1:500, Abcam, Cambridge, UK) antibodies.
For detection, an anti-rabbit IgG HRP antibody (1:3000, Dako Cytomation, Hamburg, Germany) for GFAP and for beta Tubulin were used. Immunoreactive bands were visualized by incubation with chemiluminescence reagent (Perkin Elmer, Boston, MA, USA) and signals were detected with the Fusion SL (VWR, Darmstadt, Germany). Integrated densities were measured with ImageJ software [21 ].
For detection, an anti-rabbit IgG HRP antibody (1:3000, Dako Cytomation, Hamburg, Germany) for GFAP and for beta Tubulin were used. Immunoreactive bands were visualized by incubation with chemiluminescence reagent (Perkin Elmer, Boston, MA, USA) and signals were detected with the Fusion SL (VWR, Darmstadt, Germany). Integrated densities were measured with ImageJ software [21 ].
6
Western Blot Analysis of Cell Cycle
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Denatured samples were separated on 4-15% Mini-Protean TGX SDS-PAGE gels (Bio-rad).
Protein was transferred to methanol-activated PVDF membranes (Bio-rad) by wet transfer (1x Pierce Transfer Buffer, 10% methanol) or using high molecular weight transfer conditions for the Bio-rad TransBlot Turbo (Bio-rad). Membranes were blocked in 5% milk/TBS-T and incubated with indicated primary antibodies for 1.5h at room temperature or overnight at 4˚C. Membranes were then washed and incubated with HRP-conjugated anti-mouse/rabbit secondary antibodies (Jackson Labs) for 1h at room temperature. Proteins were detected by ECL (Pierce) or Clarity (Bio-rad) detection reagents and exposure to X-ray film (Pierce).
For analysis of cell cycle, FUCCI reporter ES cells 33 were collected by trypsinization and sorted on a FACS AriaII (BD Biosciences) into mCherry+ (G0/G1) and BFP+ (S/G2/M) cell fractions.
Cells were pelleted and lysed in RIPA buffer and clarified by centrifugation for 10 min, 13,000 g at 4˚C. Lysates from the same number of cells were used for western blotting.
Protein was transferred to methanol-activated PVDF membranes (Bio-rad) by wet transfer (1x Pierce Transfer Buffer, 10% methanol) or using high molecular weight transfer conditions for the Bio-rad TransBlot Turbo (Bio-rad). Membranes were blocked in 5% milk/TBS-T and incubated with indicated primary antibodies for 1.5h at room temperature or overnight at 4˚C. Membranes were then washed and incubated with HRP-conjugated anti-mouse/rabbit secondary antibodies (Jackson Labs) for 1h at room temperature. Proteins were detected by ECL (Pierce) or Clarity (Bio-rad) detection reagents and exposure to X-ray film (Pierce).
For analysis of cell cycle, FUCCI reporter ES cells 33 were collected by trypsinization and sorted on a FACS AriaII (BD Biosciences) into mCherry+ (G0/G1) and BFP+ (S/G2/M) cell fractions.
Cells were pelleted and lysed in RIPA buffer and clarified by centrifugation for 10 min, 13,000 g at 4˚C. Lysates from the same number of cells were used for western blotting.
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