Cells were firstly harvested by centrifugation and lysed using RIPA lysis solution (Beyotime, P0013B, Beijing, China). The extracts were then analyzed by agarose gel electrophoresis with 12% SDS-PAGE, at 80 V for 20 min, and at 120 V for 2.5 h. Bio-Rad Trans-Blot (Bio-Rad, Hercules, California, USA) was used to transfer the proteins onto the polyvinylidene difluoride membranes (Millipore, ISEQ00010, Bedford, MA, USA). Blocking was performed with PBST containing 5% BSA on ice for 4 h and the membranes were incubated with primary and secondary antibodies for 4 h and overnight, respectively. BeyoECL Star Chemiluminescence Kit (Beyotime, P0018S, Beijing, China) was used for protein band detection. Quantification of protein blots was performed using AlphaView SA software (Alpha Innotech Corporation, San Leandro, CA, USA) according to the user guide using default parameters.
Trans blot
Manufactured by Bio-Rad
Sourced in United States, United Kingdom, Spain, Italy
The Trans-Blot is a laboratory equipment used for the transfer of proteins from a gel to a membrane, a process known as Western blotting. It facilitates the efficient and consistent transfer of proteins, enabling further analysis and detection.
Automatically generated - may contain errors
Lab products found in correlation
Anti alix h 270 polyclonal abs, by Santa Cruz Biotechnology (6 mentions)
Pore size nitrocellulose membrane, by Cytiva (5 mentions)
Odyssey infrared imaging system, by LI COR (5 mentions)
Nitrocellulose membrane, by Bio-Rad (5 mentions)
Immobilon western hrp substrate, by Merck Group (4 mentions)
Phospho akt ser473, by Cell Signaling Technology (4 mentions)
Anti β actin ac 74 mab, by Merck Group (4 mentions)
Nitrocellulose membrane, by Merck Group (4 mentions)
Chemidoc mp imaging system, by Bio-Rad (4 mentions)
Polyclonal goat anti mouse immunoglobulin hrp, by Agilent Technologies (4 mentions)
Clarity western ecl blotting substrate, by Bio-Rad (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Nitrocellulose membrane, by GE Healthcare (3 mentions)
P akt, by Cell Signaling Technology (3 mentions)
Bradford assay, by Bio-Rad (3 mentions)
Immobilon p, by Merck Group (3 mentions)
Anti actin ac 74 mab, by Merck Group (3 mentions)
Chemidoc system, by Bio-Rad (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Protein assay, by Bio-Rad (3 mentions)
147 protocols using trans blot
1
Western Blot Protein Analysis Protocol
2
Western Blot Analysis of Glioma Cell Lysates
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Soluble protein lysates of sub-confluent glioma cells were obtained using lysate buffer (Medical and Biological Laboratories, Woburn, MA, USA) for 20 min on ice. The proteins (50 µg proteins) were loaded and separated by 12% polyacrylamide gel electrophoresis and then transferred onto nitrocellulose membranes (GE Healthcare, Tokyo, Japan) for 30 min at 10 V with a Bio-Rad Trans Blot (Bio-Rad Laboratories, Franklin Lakes, NJ, USA). Non-specific binding was blocked with a washing buffer (PBS/0.05% containing 1% skimmed milk) for 60 min at room temperature. The primary antibody employed for the immunoblotting was β-actin mouse mAb (cat. no. 013-24553; 1:2,000; Wako Pure Chemical Industries) which was used as a loading control. The secondary antibodies employed were anti-mouse IgG (whole molecule) peroxidase conjugate (cat. no. A4416; 1:5,000; Sigma-Aldrich, St. Louis, MO, USA) for 60 min at room temperature. The immune complex was visualized using an ECL detection system (GE Healthcare) and ImageQuant Las4000 (GE Healthcare), and then analyzed using ImageJ (National Institutes of Health, Bethesda, MD, USA). The same experiments were repeated three times to confirm reproducibility.
3
Western Blotting for Enteric Infection Serology
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Western blotting was performed using standard protocol [12 (link), 18 (link)]. OMPs were resolved via SDS-PAGE and electroblotted from the gel to a 0.45 μm pore size nitrocellulose membrane by electrophoretic transfer using Bio-Rad Transblot apparatus (Bio-Rad Laboratories, USA). The electroblotting condition was set at 100 V for 4 hours according to the manufacturer’s instruction. The nitrocellulose membrane was then blocked with 3% skim milk for 30 min at room temperature to block the non-specific protein binding sites. The blocked nitrocellulose membranes were cut into strips and incubated overnight with 1:100 dilutions of human serum as the primary antibody. Sera used in this study as follows: from patients positive for S. sonnei infection; and patients from other enteric infections due to Salmonella spp., Aeromonas hydrophila, EPEC, Salmonella Typhi and Campylobacter jejuni. After primary antibody incubation, the strips were washed with PBS-T (0.05% Tween 20) 6 times for 10 minute each. It was then incubated with rabbit anti-human IgA and IgG (Sigma, USA) conjugated with alkaline phosphatase for 2 hours at room temperature with constant shaking. The strips were washed with PBS-T as above and then developed with AP-conjugated substrate for 30 minute. The enzyme reaction was stopped by rinsing the strips with distilled water.
4
ErbB Receptor Signaling in DRG Neurons
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Western blots were carried out using lysates from dissected DRG cultures. Neurons were plated at high density in polyornithine/laminin‐coated 96‐well plates (5,000 neurons per well). The neurons were incubated overnight in medium containing 25 µM Boc‐D‐FMK (Calbiochem, UK) before treating the neurons with either 10 ng/ml NGF or 500 ng/ml NRG4. After treatment, the cells were lysed in RIPA buffer and insoluble debris was removed by centrifugation. Samples were transferred to polyvinylidene difluoride membranes using the Bio‐Rad TransBlot (Bio‐Rad, CA, USA). The membranes were blocked with 5% BSA in phosphate‐buffered saline containing 0.1% TWEEN 20. The membranes were then incubated with either anti‐phospho‐ErbB3 antibody (1:1,000, ab4791, Cell Signaling Technologies), anti‐total ErbB3 antibody (1:1,000, ab4754, Cell Signaling Technologies), anti‐phospho‐ErbB4 antibody (1:1,000, ab4757, Cell Signaling Technologies), anti‐total ErbB4 antibody (1:1,000, ab4795, Cell Signaling Technologies), or anti‐β‐III tubulin antibody (1:10,000, MAB1195, R&D, MN, USA), which were detected using a peroxidase‐linked secondary antibody (Promega, WI, USA) and the ImmunoCruz™ Western Blotting Luminol Reagent (Santa Cruz, CA, USA). Densitometry was carried out using Image Studio Lite (Li‐Cor Biosciences).
5
Western Blot Analysis of BMPR2 and ALK5
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Primary antisera of BMPR2 (LS-C178875) and ALK5 (LS-C312882) were purchased from LifeSpan BioSciences (Seattle, WA, USA). Western blot analysis was performed according to the standard protocol. The ovarian and testicular homogenates were separated on a 12% SDS–PAGE gel, and the proteins were then transferred to PVDF (methanol-activated polyvinylidene difluoride) membranes (Merck Millipore, Darmstadt, Germany) using Bio-Rad trans-blot (Bio-Rad, Hercules, CA, USA). The membranes were incubated overnight in 5% nonfat milk powder in 10 mM PBST buffer at 4 °C, blocked and incubated again with the primary antiserum (1:800 dilution) diluted with 5% nonfat milk powder in 10 mM PBST, at 25 °C for 2 h. The membrane was subjected to three 5 min washes with PBST and incubated in horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Boster, Wuhan, China; 1:500 dilution) diluted with 5% nonfat milk powder in 10 mM PBST for 1 h at 25 °C. According to the protocol recommended by the manufacturer of the ECL kit (MXB, Fuzhou, China), the membranes were washed with PBST and detected using Bio-Rad ChemiDocTM MP (Bio-Rad, Hercules, CA, USA).
6
Western Blot Analysis of Liver Proteins
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Western blot analysis was performed as previously reported [62 (link)]. Briefly, the liver was homogenized at 4 °C in RIPA buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton-X100, 1× protease inhibitor) and total protein lysates, obtained by centrifugation at 14,000× g for 15 min at 4 °C, were subjected to SDS-PAGE and transferred onto a PVDF membrane (IPVH00010 Millipore) using a Bio-Rad Transblot (Bio-Rad, Milan, Italy). Membranes were blocked at room temperature in BSA (3% w/v bovine serum albumin, 0.3% v/v Tween-20, in PBS) and probed with anti-phospho-AMPKα (1:1000) and AMPK antibodies (1:1000) (Cell Signaling, Danvers, MA, USA). The images were acquired using a Biorad Gel Doc XR System.
7
Quantitative Cytokeratin Immunoblotting
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Defined amounts (70 μg) of proteins from soluble cellular extracts were loaded and separated on 10% SDS-PAGE and electrotransferred onto a nitrocellulose membrane (GE Healthcare Life Sciences, United Kingdom) using a Bio-Rad Transblot (Bio-Rad). Proteins were visualized by reversible staining with Ponceau-S solution and destained in PBS. Membranes were blocked at room temperature in milk buffer (1 × PBS, 5–10 g/100 mL non fat dry milk, 0.2% g/100 mL Tween-20) and then incubated at 4°C overnight with 1:250 monoclonal anti-pan cytokeratin antibodies (mixture) (Sigma-Aldrich, Milan, Italy), which recognize the following human cytokeratins, according to their molecular weight: K1 (68 kDa), K4 (59 kDa), K5 (58 kDa), K6/K10 (56 kDa), K13 (54 kDa), K8 (52 kDa), K18 (45 kDa), and K19 (40 kDa). As a secondary antibody, a goat anti-mouse IgG+IgM (1:5000; Jackson ImmunoResearch Laboratories, Baltimore Pike, West Grove, PA, USA) was used. The immunocomplexes were visualized by the ECL chemiluminescence method (ECL, Amersham Biosciences, United Kingdom) and analyzed by an imaging system (ImageQuant™ 400; GE Healthcare Life Sciences). Densitometric analyses were conducted using the GS-800 imaging densitometer (Bio-Rad). The GAPDH antibody (Sigma-Aldrich) was used to normalize the results.
8
Western Blot Protein Analysis
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Extracted proteins (30 μg/sample) were denatured, separated using SDS-PAGE (4 to 12 % gradient gel at 150 V for 2•5-3 h) and transferred to a nitrocellulose membrane overnight (approximately 16 h) at 20 V using the Bio-Rad Transblot (Bio-Rad). Membranes were blocked in 5 % fat-free milk in 20 mmol/l TRIS, 150 mmol/l NaCl, pH 7•5, and 0•1 % Tween-20 (TBST) for 3 h and then incubated with a primary antibody (i.e. anti-β-actin (ACTB) (1:2000; #4970), anti-transferrin (TF) (1:2000; ab82411), anti-glucose-regulated protein (GRP78) (1:1000; #3183) or antialbumin (ALB) (1:1000; #4929)) at 4°C overnight with gentle rocking. After washing three times with TBST, the membranes were incubated for 2 h with a secondary antibody (horseradishperoxidase-linked anti-rabbit IgG) at 1:10 000 dilution. The membranes were then washed with TBST, followed by development using enhanced chemiluminescence detection (SuperSignal West Pico) according to the manufacturer's instructions. Western blots were quantified by measuring the intensity of light emitted from correctly sized bands under ultraviolet light using a ChemiDoc EQ system and Quantity One software (Bio-Rad). All the antibodies were purchased from Cell Signaling Technology, except for TF (ab82411; Sigma-Aldrich). Multiple exposures of each Western blot were performed to ensure linearity of chemiluminescence signals.
9
Protein Quantification and Western Blot Analysis
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Cells were collected, and total protein was extracted. BCA solution (FUDE Biology, Hangzhou) was prepared to detect protein concentration of the samples to be tested. The prepared protein samples were taken out, dissolved on the ice box, and then, added into the electrophoresis tank for 40 min. The PVDF membrane of corresponding size was cut, and the separation glue was closely attached to the PVDF membrane, and the protein was transferred to the PVDF membrane in Bio-Rad Trans-Blot. After membrane transfer, remove PVDF membrane, wash it for 3 times, and add an appropriate amount of 5% skim milk powder for 1 h. The membrane was washed with TBS-T for 3 times, and the corresponding strip was added into the primary antibody solution and incubated overnight at 4°C. The next day, the strips were washed with TBS-T for 3 times and then added with corresponding secondary antibodies and incubated at 4°C for 4 h. ECL luminescent solution (Omiget Pharmaceutical Technology Co., Ltd., Beijing) was prepared and uniformly dropped on PVDF membrane, and the reaction was carried out in the darkroom for several seconds. The cut film is completely covered on PVDF membrane, exposed for 5-10 seconds, then put into the prepared developer, using IPP6.0 software for gray value analysis of the film.
10
Quantitative Analysis of Atrial Proteins
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Whole-cell lysates from frozen atrial tissue were run simultaneously to ensure identical conditions. From each sample, 60 mg total protein was loaded on a 4% to 12% gradient sodium dodecyl sulfate polyacrylamide electrophoresis gel (Life Technologies, Grand Island, NY). The proteins were transferred to activated polyvinylidene difluoride membranes (Millipore, Billerica, MA) with a semidry transfer cell apparatus (Trans-Blot; Bio-Rad, Hercules, CA). Each membrane was incubated with specific primary antibodies overnight at 4 C and secondary antibodies for 1 hour at room temperature. Glyceraldehyde-3-phosphate dehydrogenase (Cell Signaling Technology, Danvers, MA) was used to normalize the protein loading.
JAK2, suppressor of cytokine signaling 3 (SOCS3), p-STAT1/3/5, STAT1/3/5, apoptotic markers p-Bc12, Bc12, caspase 3, Mcl-1, Bcl-xL, and p-Akt (Cell Signaling Technology), and oxidative stress markers nuclear factor (NF)-kB (Abcam, Cambridge, United Kingdom) and manganese-containing superoxide dismutase (R&D Systems, Minneapolis, MN) were probed. Neurotrophin-3 (Abbiotec, San Diego, CA) and nerve growth factor (Santa Cruz Biotechnology) were examined as markers for neurogenesis. Immune complexes were visualized with an enhanced chemiluminescence detection system (Thermo Scientific, Amersham, Piscataway, NJ).
JAK2, suppressor of cytokine signaling 3 (SOCS3), p-STAT1/3/5, STAT1/3/5, apoptotic markers p-Bc12, Bc12, caspase 3, Mcl-1, Bcl-xL, and p-Akt (Cell Signaling Technology), and oxidative stress markers nuclear factor (NF)-kB (Abcam, Cambridge, United Kingdom) and manganese-containing superoxide dismutase (R&D Systems, Minneapolis, MN) were probed. Neurotrophin-3 (Abbiotec, San Diego, CA) and nerve growth factor (Santa Cruz Biotechnology) were examined as markers for neurogenesis. Immune complexes were visualized with an enhanced chemiluminescence detection system (Thermo Scientific, Amersham, Piscataway, NJ).
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