This procedure was performed as in our previous report.50 (link) Protein extracts from cells were prepared in RIPA buffer (Beyotime) containing 1% NP-40 (Beyotime) and a cocktail of protease and phosphatase inhibitors (Beyotime). Protein concentrations were determined using the Bio-Rad protein assay (Bio- Rad, Hercules, CA, USA; 500-0006). Thirty micrograms of total protein were loaded onto 8-10% SDS-PAGE gels and transferred onto a PVDF membrane using a Trans-Blot Turbo SystemTM (Bio-Rad) and Transfer packTM (Bio-Rad; 1704156). The primary antibodies used for the analysis were as follows: Tuj1 (#ab18207; 1:1000; Abcam, Cambridge, UK), GFAP (ab7260; 1:1000; Abcam), LC3 (#ab128025; 1:500; Abcam), Beclin-1(#ab302669; 1:1000; Abcam), p62 (#ab207305;1:1000; Abcam), PPARγ (#ab178860; 1: 1000; Abcam), and GAPDH (1:1000; Beyotime). Protein bands were visualized using HRP-linked secondary antibodies (Bio-Rad, anti-mouse 1706516, anti-rabbit 1706515) and the Clarity Western ECL substrate (Bio-Rad, 1705061) with a ChemiDoc MP imaging system (Bio-Rad). The blots were stripped with glycine (0.2 M, pH 2.5) stripping buffer and reprobed with the appropriate antibodies. The intensity of each band was detected and measured by Image J software (version 1.4; National Institutes of Health, Bethesda, MD, USA; http://imagej.nih.gov/ij/ ).
Trans blot turbo system
Manufactured by Bio-Rad
Sourced in United States, Germany, United Kingdom, Italy, France, Singapore, Australia, Canada
The Trans-Blot Turbo system is a rapid protein transfer device designed for the transfer of proteins from polyacrylamide gels to membranes. The system utilizes a unique blotting technology to enable fast and efficient transfer of proteins, reducing the transfer time compared to traditional methods.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc mp imaging system, by Bio-Rad (49 mentions)
Clarity western ecl substrate, by Bio-Rad (46 mentions)
Ripa buffer, by Thermo Fisher Scientific (39 mentions)
Nitrocellulose membrane, by Bio-Rad (38 mentions)
Image lab software, by Bio-Rad (34 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (28 mentions)
Mini protean tgx gel, by Bio-Rad (26 mentions)
Chemidoc imaging system, by Bio-Rad (26 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (25 mentions)
Laemmli sample buffer, by Bio-Rad (23 mentions)
Pvdf membrane, by Bio-Rad (22 mentions)
Ripa buffer, by Merck Group (19 mentions)
Dc protein assay, by Bio-Rad (16 mentions)
Westernbright ecl, by Advansta (16 mentions)
Chemidoc touch imaging system, by Bio-Rad (15 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (15 mentions)
Chemidoc xrs system, by Bio-Rad (15 mentions)
Odyssey imaging system, by LI COR (14 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (13 mentions)
Chemidoc mp system, by Bio-Rad (13 mentions)
679 protocols using trans blot turbo system
1
Western Blot Analysis of Protein Expression
2
Comprehensive Western Blot Analysis Protocol
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TA muscles were homogenized with Ultra-Turrax (Ika Werke, Staufen, Germany) in a lysis buffer containing 20 mM Tris-HCl (pH 7.4), 10 mM EGTA, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1% Sodium Dodecyl Sulfate (SDS) supplemented with protease and phosphatase inhibitors. Protein extracts from cells were performed in RIPA buffer (10 mM Tris-Cl (pH 8.0), 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS) supplemented with protease and phosphatase inhibitors. Proteins were quantified using a BCA protein assay kit (Pierce, Rockford, IL, USA) following the standard protocol for western blotting. Thirty to fifty µg of total protein were loaded on 4–20 % polyacrylamide precast gels (Criterion TGX Stain-free precast gels; Bio-Rad). Proteins were transferred onto a nitrocellulose membrane using a Trans-Blot Turbo SystemTM (7 min at 2.5 A) and Transfer packTM (Bio-Rad). Primary antibodies used to probe membranes are indicated in Supplementary Table S1 . After the incubation with the appropriate horseradish-peroxidase-conjugated secondary antibody [58 (link)], bands were visualized using the Clarity Western ECL substrate with a ChemiDoc MP imaging system (Bio-Rad). Bands were quantified for densitometry using the Bio-Rad Image Lab software. Uncropped western blots are provided in Supplementary Material.
3
Immunoblotting Assay for CUDC-907 Effects
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Immunoblotting assays were performed by treating NB cells with different concentrations of CUDC-907 for 6 h, followed by cell lysis using RIPA buffer (89900; Thermo Fisher Scientific) supplemented with protease inhibitor cocktail (Complete mini EDTA free, Roche, Basel, Switzerland) and phosphatase inhibitor cocktail (PhosSTOP, Roche). Protein samples were quantified with Bradford assay (5000205; Bio-Rad, Hercules, CA, USA) and equal amounts of total protein were separated on 4–12% SDS-PAGE gels, followed by transfer to PVDF membranes using a Bio-Rad Trans-Blot Turbo TM system, then blocking with 5% BSA solution, and probing with corresponding primary antibodies (1:1000 dilution) overnight at 4 °C [25 (link)]. This was followed by washing and incubating the membranes with either anti-mouse or anti-rabbit IgG HRP-conjugated secondary antibody (1:10,000 dilution) for 2 h. Protein bands on membranes were developed using Clarity ECL Western substrate (Bio-Rad) and visualized using the ChemiDoc XRS Plus system (Bio-Rad). Densitometric analysis of the protein bands was performed using the ImageJ software.
4
Tyro3 Protein Isolation and Western Blot
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Protein isolation from cells was performed using RIPA lysis buffer containing 1x protease inhibitor cocktail Complete (Roche, Basel, Switzerland), 1 mM PMSF, and 1 mM orthovanadate (Sigma-Aldrich, St. Louis, MO, USA). The protein concentration was determined using DC™ Protein Assay (Bio-Rad, Feldkirchen, Germany). Western blots were performed using precast Mini Protein TGX gels and the semi-dry Trans-Blot TurboTM System (Bio-Rad, Germany). Antibodies and related secondary antibodies (DAKO, Tokyo, Japan) were used at a dilution of 1:1000 in TBST for Anti-Tyro3 (Cell Signalling, Danvers, MA, USA, #5585). Anti-GADPH (Cell Signalling, USA, #5174) was used as a loading control.
5
Western Blot Protein Analysis
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Cells were lysed in radioimmunoprecipitation assay lysis buffer supplemented with PMSF, protease and phosphatase inhibitors at 4°C for 30 minutes, followed by centrifugation at 12 000 g for 15 minutes. Protein concentration was determined using the BCA protein assay kit. Protein samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to NC membranes using the Trans‐Blot® TurboTM system (Bio‐Rad). Nitrocellulose membranes were blocked in TBST supplemented with 5% nonfat milk and incubated with primary antibodies for 8 hours at 4°C. The NC membranes were washed four times with TBST and incubated with fluorescence secondary antibodies for 1.5 hours at RT. Immunogenicity was tested using an Odyssey v3.0 image scanner (LI‐COR Biosciences). Band quantification was analyzed by ImageJ software.
6
Western Blot Analysis of Myoblast Proteins
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Whole protein extracts were generated from myoblast cell cultures using an ultrasonic sonicator with a MS73 tip (Bandelin Sonopuls). The proteins were separated by SDS gel electrophoresis using 4–15% TGX gels (BioRad #456–8087). Western blotting was performed using the Trans-Blot®; TurboTM system (BioRad). Proteins were transferred to low fluorescent PVDF membranes (part of Trans-Blot®; TurboTM RTA Transfer Kit #170-4274). Membranes were blocked with 5% BSA or 5% skim milk in 1xTBS/0, 1% Tween®; 20. Following primary antibodies were used: lamin A/C 4A7 (provided by Glenn E. Morris), lamin B1 (D4Q4Z, Cell Signaling) p16INK4A (ab108349, Abcam), p21 (Cell Signaling #2947). For quantification mouse antiGAPDH (Milipore #MAB374) or rabbit antiGAPDH (Cell Signaling GAPDH (D16H11) XP #5174) were used. As secondary antibodies we used donkey anti-mouse IRDye 680RD, donkey anti-mouse IRDye 800CW, donkey anti-rabbit IRDye 680RD and donkey anti-rabbit IRDye 800 CW. All western blot images were obtained using a Licor FC. Quantification was done using the Licor ImageStudio Software. Western blots were repeated at least two times to confirm the results.
7
Western Blot Analysis of Protein Levels
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The protein expression levels of PLK1, PGP and HGS were analysed by a Western blot. Equal amount of protein from each sample was loaded onto SDS‐PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) gel, separated by electrophoresis and transferred onto a polyvinylidene difluoride (PVDF) membrane by a semidry Western blot system (Trans‐Blot® TurboTM System, Bio‐Rad, Singapore). The membrane was then blocked in 5% non‐fat milk for 2 hours at 37°C and incubated at 4 ℃ with primary antibodies for 16‐18 hours Membranes were washed three times using TBST buffer (a mixture reagent of TBS [Tris‐buffered saline] and Tween‐20) and then incubated with secondary antibodies in TBST buffer for 2 hours at 37°C. Bands were visualized with an alkaline phosphatase detection kit (C3206, Beyotime Biotechnology Inc). The following commercially available antibodies were used as primary antibodies: (anti‐RIMS3 antibody, anti‐CPI‐17, anti‐metabotropic glutamate receptor 2 antibody, anti‐superoxide dismutase 1 antibody, anti‐PP2A beta antibody, anti‐beta‐actin antibody).
8
Western Blot Analysis of Protein Extracts
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Proteins were extracted from plants as described by Han et al. (2022) (link). Western blots were performed by SDS-PAGE with the Mini-PROTEA§ Tetra System (BIO-RAD). Proteins were transferred to the polyvinylidene fluoride (PVDF) membrane with the TransBlot turboTM system (Bio-rad), and the membrane was blocked for 2 h with 1 × Tris-buffered saline (TBS) containing skim milk (0.5%) followed by 2 h incubation with primary antibody. After washing with TBS, the membrane was incubated with the secondary antibody for 1 h, followed by wash with TBS. The photographic developer was sprayed (A solution + B solution mix at 1:1) onto the membrane. The primary antibody was Anti-DYKDDDDK Monoclonal Antibody (TRANS), Actin mouse anti-antibody (Bioss), and the secondary antibody was Goat Anti-Mouse IgG, HRP (TRANS). The blot was recorded using Tanon 5200 Multi.
9
Quantification of Leptin Receptor Protein
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The total protein concentration of the tissue homogenate from cultured nasal fibroblasts was measured using a Bicinchoninic acid Protein Assay Kit per the manufacturer’s instructions (Thermo Fisher Scientific), and 20 ng of protein was loaded for electrophoresis on a Mini-PROTEAN® TGXTM Gel (Bio-Rad Laboratories, Hercules, CA, United States). The protein was then transblotted using a Trans-Blot® TurboTM Transfer Pack and Trans-Blot® TurboTM system (Bio-Rad Laboratories). Following transfer, the blots were blocked with 5% non-fat dry milk in 0.1% Tween 20/Tris-buffer saline (TBS) and TBS alone, and then incubated with anti-leptin receptor antibody (1:100; Novus Biologicals, Centennial, CO, United States). They were then incubated with a horse radish peroxidase-conjugated (HRP-conjugated) anti-rabbit IgG antibody (Dako; Agilent Technologies, Santa Clara, CA, United States). Subsequently, the blots were developed using chemiluminescent Western blot detection reagents (Amersham ECL Prime; GE Healthcare, Chicago, IL, United States) according to the manufacturer’s instructions. The blot density was scanned using Fusion Solo S (Vilber, Marne-la-Vallée cedex, France).
10
Protein Isolation and Western Blot Analysis
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Protein isolation from cells and fresh frozen tumor samples were performed using RIPA lysis buffer containing 1x protease inhibitor cocktail cOmplete (Roche, Switzerland), 1mM PMSF, and 1mM orthovanadate (Sigma-Aldrich, USA). Protein concentration was determined using DC™ Protein Assay (Bio-Rad, Germany). Western blots were performed using precast Mini Protein TGX gels and the semi-dry Trans-Blot TurboTM System (Bio-Rad, Germany). Antibodies used for specific gene detection are shown in Additional file: Table S2 .
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