The cells were lysed in Cell Lysis Buffer (Cell Signaling Technology, Delaware, CA, USA) containing a 1x Protease/Phosphatase Inhibitor Cocktail (Cell Signaling Technology). The protein content was measured with a protein assay kit (Pierce, Hercules, CA, USA). Protein samples (15 μg), together with a protein marker (Precision Plus Protein Western C Standards; Bio-Rad), were separated on 12% Mini-Protean TGX gels (Bio-Rad, Richmond, CA, USA) for 30 min at 200 V. The separated gels were transferred to a polyvinylidene fluoride (PVDF) membrane for 3 min using the Trans-Blot Turbo Transfer system (Bio-Rad) with Trans-Blot Transfer Packs. Western blots with Runx2), ALP, OSX, and β-actin (ACTB) were processed on the iBind Western System (Life Technologies, Carlsbad, CA, USA) as specified by the antibody manufacturer (anti- Runx2) [Abcam, Tokyo, Japan], anti-ALP [Abcam], anti-Sp7/osterix [OSX; Abcam], and anti- ACTB [Bio-Rad] primary antibodies; and horseradish peroxidase-conjugated anti-mouse secondary antibody [Bio-Rad]). The incubated membranes were developed using an enhanced chemiluminescence system (SignalFire Plus ECL Reagent; Cell Signaling Technology). The band density was quantified using the NIH-Image J software and normalized to that of ACTB on day 0.
Trans blot transfer pack
Manufactured by Bio-Rad
Sourced in United States
The Trans-Blot Transfer Packs are a versatile and efficient solution for protein transfer in Western blotting applications. These packs provide the necessary components for the transfer of proteins from a gel to a membrane, enabling the detection and analysis of specific proteins. The packs include pre-cut transfer membranes, filter papers, and fiber pads, ensuring a streamlined and consistent transfer process.
Automatically generated - may contain errors
Lab products found in correlation
Trans blot turbo transfer system, by Bio-Rad (5 mentions)
Ibind western system, by Thermo Fisher Scientific (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Bca assay, by Thermo Fisher Scientific (2 mentions)
Rabbit anti lc3b, by Cell Signaling Technology (2 mentions)
Rabbit anti beclin 1, by Cell Signaling Technology (2 mentions)
Precision plus protein westernc standard, by Bio-Rad (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
Protease phosphatase inhibitor cocktail, by Cell Signaling Technology (1 mentions)
Anti runx2, by Abcam (1 mentions)
Horseradish peroxidase conjugated anti mouse secondary antibody, by Bio-Rad (1 mentions)
Signalfire plus ecl reagent, by Cell Signaling Technology (1 mentions)
Anti alp, by Abcam (1 mentions)
Clarity western ecl substrate kit, by Bio-Rad (1 mentions)
0.2 μm pore size nitrocellulose membrane, by Bio-Rad (1 mentions)
Hrp labeling kit, by Dojindo Laboratories (1 mentions)
Bio safe coomassie stain, by Bio-Rad (1 mentions)
Precision plus protein dual color standard, by Bio-Rad (1 mentions)
2 mercaptoethanol 2 me, by Fujifilm (1 mentions)
7 protocols using trans blot transfer pack
1
Western Blot Analysis of Osteogenic Markers
2
Protein Expression Analysis Protocol
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Both exfoliative and cultured cells were lysed in Cell Lysis Buffer containing protease and phosphatase inhibitor (1× Protease/Phosphatase Inhibitor Cocktail). Protein concentration was measured with a protein assay kit. Equal amounts (15 μg) of protein along with a protein marker (Precision Plus Protein Western C Standards) were separated on Mini-protean TGX gels for 30 min at 200 V. The separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane using the Trans-Blot Turbo Transfer system (Bio-Rad) with Trans-Blot Transfer Packs. Western blots were processed on the iBind Western System (Life Technologies, Carlsbad, CA, USA) with primary antibodies and horseradish peroxidase-conjugated secondary antibodies. The protein bands were developed using an enhanced chemiluminescence system (SignalFire Plus ECL Reagent). Band density was quantified using the software NIH-Image J.
3
Enzyme-Linked Immunosorbent Assay Development
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SO and CHA powders were purchased from Sigma-Aldrich Co., LLC (Tokyo, Japan). Horseradish peroxidase (HRP)-labeled goat anti-rabbit polyclonal IgG (H + L) antibodies were obtained from Funakoshi Co., Ltd. (Tokyo, Japan). Commercial serum and urine samples of healthy volunteers were obtained from Cosmo Bio Co., Ltd. (Tokyo, Japan). Bio-Safe Coomassie stain, Clarity Western ECL substrate kits, Precision Plus Protein Dual Color Standards, Trans-Blot Transfer Packs, and 0.2 μm pore-size nitrocellulose membranes were obtained from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). A 96-well ELISA plate H was purchased from Sumitomo Bakelite Co., Ltd. (Tokyo, Japan). An HRP labeling kit was obtained from Dojindo Laboratories (Kumamoto, Japan). Blocking One and dimethyl sulfoxide (DMSO) were purchased from Nacalai Tesque, Inc (Kyoto, Japan). The Prominence LC-2010 HPLC system and the reversed-phase chromatography (RPC) column Shim-pack GIST C18-AQ were purchased from Shimadzu Corporation (Kyoto, Japan). Freund’s complete adjuvant (FCA), Freund’s incomplete adjuvant (FIA), o-phenylenediamine dihydrochloride (OPD) tablets, 2-mercaptoethanol (2-ME), and all other reagents were obtained from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan) and Wakenyaku Co., Ltd. (Kyoto, Japan).
4
Quantitative Protein Extraction and Analysis
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Total cell protein was extracted utilising a modified radio immunoprecipitation (RIPA) buffer consisting of 20 mM Tris-HCl (pH 7.4), 137 mM NaCl, 10% Nonidet-P40 and 10% Na-deoxycholate, 42 µL complete EDTA-free protease inhibitor tablet solution, 1 mM PMSF, 1 mM Na3VO4 and 1 mM NaF phosphatase inhibitors. Protein concentration was determined using a Bradford Assay25 (link). Cell lysates were diluted in Laemmeli sample buffer, boiled for 5 min and 50 µg protein was separated by 12% SDS-PAGE-gel electrophoresis (Bio Rad Mini-Protean ®TGX™ fast cast system). Proteins were transferred onto a PVDF (poly vinydilene difluoride) membrane using BIO-RAD Trans-Blot transfer packs and a BIO-RAD Trans-Blot turbo transfer system. Membranes were blocked for 60 minutes in 5% non-fat milk made up in 1 × TBS-T (Tris-buffered saline and 1% Tween20), followed by incubation with appropriate primary and secondary anti-bodies. Band intensities were detected with a BIO-RAD Chemidoc MP imaging system using Image Lab software (version 4.1) and expressed as a percentage relative to band intensities of untreated control cells. β-actin was used as a loading control for all membranes.
5
Yeast Protein Extraction and Western Blotting
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Yeast transformants in the yLFM-P21-5′ and yLFM-PERP strains were grown 8 h in selective medium containing 0.128% galactose and collected by centrifugation. Cells were resuspended in 100 µl distilled water, and 100 µl 0.2 M NaOH were then added. The mixture was then incubated for 5 min at room temperature, pelleted, resuspended in 50 µl SDS sample buffer (0.06 M Tris-HCl, pH 6.8, 5% glycerol, 2% SDS, 4% β-mercaptoethanol, 0.0025% bromophenol blue), boiled for 3 min and pelleted again (47 (link)). Ten microliters of yeast supernatant were resolved on 4–15% Mini-Protean TGX gels and transferred to nitrocellulose membranes by Trans-Blot Turbo Blotting System using the Trans-Blot Transfer Pack, (Bio-Rad, Hercules, CA, USA). A specific antibody directed against P63 (clone 4A4, sc-8431; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was diluted in 1% non-fat dry milk dissolved in PBS-T. Phosphoglycerate kinase 1 (PGK1) was used as a loading control (clone 22C5D8; Life Technologies; Thermo Fisher Scientific, Inc., Waltham, MA, USA). After washing, the blots were incubated with the appropriate IgG horseradish peroxidase-conjugated secondary antibody (anti-mouse IgG HRP, A9044; Sigma-Aldrich, St. Louis, MO, USA), and immune complexes were visualized with ECL Fast Pico (Immunological Sciences, Rome, Italy). Chemiluminescence was analyzed by Alliance LD (Uvitec, Cambridge, UK).
6
Western Blotting of Cell Signaling Proteins
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Cells were harvested by trypsinization, and lysis was performed using RIPA buffer. The protein concentration was determined with a BCA assay (Pierce Biotechnology, 23225, Rockford, IL, USA). Equal amounts of protein were separated on SDS–PAGE gels. Then, the proteins on the gels were transferred to a polyvinyl difluoride membrane (Trans-Blot® Transfer Pack, Bio–Rad 1704157, Hercules, CA, USA) with a semidry blotting technique (Trans-Blot® Turbo™ Transfer System, Bio–Rad 1704150, Hercules, CA, USA). The membranes were blocked in 5% PBS-BSA. The following antibodies were used for Western blotting: rabbit anti-BECLIN 1 (Cell Signaling, 3738S, Danvers, MA, USA), anti-β-ACTIN (Sigma, A5441, Burlington, MA, USA), rabbit anti-LC3B (Cell Signaling, 2775, Danvers, MA, USA), anti-OCT2 (ThermoFisher, ACT-020, Waltham, MA, USA), rabbit anti-GRP78 (Abcam, ab 21685, Cambridge, UK), rabbit anti-CHOP (Cell signaling, 5554, Danvers, MA, USA) and anti-CASPASE 12 (Cell signalling, 2202, Danvers, MA, USA). Densitometry was performed using LabImage 1D software version L320 (Kapelan Bio-Imaging, Leipzig, Germany).
7
Glomerular Protein Extraction and Western Blot
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Glomeruli were isolated by perfusion with DynabeadsTM and were subjected to glass-glass homogenization in lysis buffer (RIPA buffer) [52 (link)]. Cells were harvested by trypsinization, and lysis was performed using RIPA buffer. The protein concentration was determined with a BCA assay (Pierce Biotechnology, Waltham, MA, USA, 23225). Equal amounts of protein were separated on SDS–PAGE gels. Then, the proteins on the gels were transferred to a polyvinyl difluoride membrane (Trans-Blot® Transfer Pack, Bio-Rad, Hercules, CA, USA, 1704157) with a semidry blotting technique (Trans-Blot® Turbo™ Transfer System, Bio-Rad, Hercules, CA, USA, 1704150). The membranes were blocked in 5% PBS-BSA. The following antibodies were used for Western blotting: rabbit anti-BECLIN 1 (Cell Signaling, Danvers, MA, USA, 3738S), rabbit anti-VEGF (Abcam, Cambridge, UK, 51745), anti-β-actin (Sigma, St. Louis, MI, USA, A5441), rabbit anti-LC3B (Cell Signaling, Danvers, MA, USA, 2775), rabbit anti-β-COP (Abcam, Cambridge, UK, 2899), goat anti-SEC23 (Abcam, Cambridge, UK, 99552), and rabbit anti-RAB5 (Abcam, Cambridge, UK, 218624). Densitometry was performed using LabImage ID software (Kapelan Bio-Imaging).
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