Cell sample of 15 ml culture was pelleted by centrifugation and re-suspended in 150 μl TBST buffer (50 mM Tris–HCL, 100mM NaCl, 0.1% Tween-20, pH7.6). Sonication of the cell suspension gave total cell extracts, which were fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein bands on the gel were transferred onto a nitrocellulose membrane using a Trans-Blot Semi-Dry Transfer Cell (Bio-Rad). For immunoblotting, the membrane was immerged in 5% skim milk blocking agent, then incubated with individual primary antibodies and finally with corresponding second antibodies. TFB3 antisera were raised against S. solfataricus TFB3 (18 (link)) (kindly provided by Prof. Malcolm F. White) whereas PCNA3 antisera against S. islandicus PCNA3 were reported previously. Secondary antibodies were purchased from Thermo Fisher Scientific, and hybridization signals were detected using the ECL western blot substrate (Thermo Fisher Scientific), and visualized by exposure of the membrane to an X-ray film (Agfa HealthCare, Belgium).
Trans blot semi dry transfer cell
Manufactured by Bio-Rad
Sourced in United States, Germany
The Trans-Blot Semi-Dry Transfer Cell is a laboratory equipment designed for the efficient transfer of proteins from polyacrylamide gels to membranes. It utilizes a semi-dry blotting method to enable the transfer of proteins from gels to membranes for further analysis and detection.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (10 mentions)
Pvdf membrane, by Merck Group (8 mentions)
Ripa buffer, by Thermo Fisher Scientific (6 mentions)
Pvdf membrane, by Bio-Rad (4 mentions)
Hsp90, by Cell Signaling Technology (4 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (4 mentions)
Nitrocellulose membrane, by Bio-Rad (4 mentions)
Odyssey blocking buffer, by LI COR (3 mentions)
Amersham hybond ecl, by GE Healthcare (3 mentions)
Image lab software, by Bio-Rad (3 mentions)
Murine anti his antibody, by Qiagen (3 mentions)
Chemiluminescence reagent, by New England Biolabs (3 mentions)
Chemidoc mp system, by Bio-Rad (3 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (3 mentions)
Laemmli sample buffer, by Bio-Rad (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Las4000 mini lumino image analyzer, by GE Healthcare (2 mentions)
Dc protein assay reagent, by Bio-Rad (2 mentions)
Irdye 800cw goat anti rabbit, by LI COR (2 mentions)
Irdye 680rd donkey anti mouse, by LI COR (2 mentions)
93 protocols using trans blot semi dry transfer cell
1
Immunoblotting of Thermophilic Archaeal Proteins
2
Protein Extraction and Western Blotting
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Small pieces of frozen tissues were placed into 1.5 mL centrifuge tubes and then homogenized by using a Turrax homogenizer (IKA, Germany) in lysis buffer for protein extraction (10 mM Tris/HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM sodium orhtovanadate, 0.5% Triton X-100, 1mM PMSF, protease inhibitor cocktail). Tissues lysates were obtained by centrifugation at 14.000g for 20 min at 4°C.
Equal amount of protein were subjected to 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride transfer membrane (Immobilon-P transfer membrane, Millipore, MA) using a Trans-Blot semi-dry transfer cell (BioRad, CA, USA). Blots were incubated with the appropriate antibody (anti-SERPINE2 1:1000, R&D Systems, MN, USA; anti-ITIH5 1:1000, ABCAM, UK; anti-WISP2 1:1000, Abnova, Taiwan). Immunoblots were visualized with Immobilon Western Detection kit (Millipore, MA) using horseradish peroxidase-labeled secondary antibody. To confirm equal loading for each sample, membranes were incubated with stripping buffer (100 mM β-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl pH 6.7) and re-blotted with anti-β-actin antibody (Sigma, MO, USA). Images were captured and analyzed with an EC3 imaging system (UVP, CA, USA).
Equal amount of protein were subjected to 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride transfer membrane (Immobilon-P transfer membrane, Millipore, MA) using a Trans-Blot semi-dry transfer cell (BioRad, CA, USA). Blots were incubated with the appropriate antibody (anti-SERPINE2 1:1000, R&D Systems, MN, USA; anti-ITIH5 1:1000, ABCAM, UK; anti-WISP2 1:1000, Abnova, Taiwan). Immunoblots were visualized with Immobilon Western Detection kit (Millipore, MA) using horseradish peroxidase-labeled secondary antibody. To confirm equal loading for each sample, membranes were incubated with stripping buffer (100 mM β-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl pH 6.7) and re-blotted with anti-β-actin antibody (Sigma, MO, USA). Images were captured and analyzed with an EC3 imaging system (UVP, CA, USA).
3
Western Blot Analysis of Bacterial Proteins
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Bacteria grown overnight in TGP containing 0.2 mM KH2PO4 (limited Pi concentration) were centrifuged, and a culture volume corresponding to an OD600 of 1.0 (approx. 109 cells) was resuspended in 0.1 ml Application Buffer (0.5 M Tris/HCl, 2% SDS, 5% 2-mercaptoethanol, 10%, v/v, glycerol and 0.01% bromophenol blue) and boiled for 5 min. Ten μl samples were resolved by standard SDS-PAGE (12.5% acrylamide). Following electrophoresis, proteins were transferred to a nitrocellulose membrane using a Trans-blot Semi-Dry Transfer Cell (BioRad, USA), as recommended by the manufacturer. The membrane was subjected to blocking with 5% skimmed milk and exposed to anti-FLAG M2 (Sigma) monoclonal antibodies, anti-RpoS (Neoclone) monoclonal antibodies (1,000X dilution) or anti-RpoD (Santa Cruz) monoclonal antibodies (5,000X dilution), followed by exposure to anti-mouse IgG serum conjugated to peroxidase (Thermo Scientific) diluted 10,000-20,000. Membranes were developed using the Clarity Max detection kit (Bio-Rad) and read in the Bio-Rad Imaging System.
4
SDS-PAGE and Western Blot Analysis
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Ten milliliters of NQO/UV-treated or reference cultures were collected by centrifugation and resuspended in 150 μl of TBST buffer (50 mM of Tris–HCl, 100 mM of NaCl, and 0.1% Tween-20, pH 7.6). Cells in the cell suspensions were then disrupted by sonication using a JY92-IIN ultrasonic homogenizer (Scientz Biotechnology), giving cell extracts for each cell sample. Proteins in each sample (10 μg) were separated according to their sizes on a 10% gel by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Proteins in the gel were then transferred onto a nitrocellulose membrane using a Trans-Blot Semi-Dry Transfer Cell (Bio-Rad). For immunoblotting, the membrane was first immersed in 5% skim milk blocking agent for 60 min and then incubated with individual primary antibody for another 60 min and finally with corresponding secondary antibody for 60 min. Hybridization signals were generated on the membrane using the ECL western blot substrate (Millipore) and visualized by exposure of the membrane to an X-ray film.
5
Western Blot Analysis of Protein Samples
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The cells were lysed with RIPA lysis buffer in the presence of the protease and phosphatase inhibitor cocktail (Sigma). Individual proteins in the samples were separated via SDS-PAGE and transferred onto a PVDE membrane (Bio-Rad, Hercules, CA, USA) using a Trans-Blot semi-dry transfer cell (Bio-Rad) with buffer containing 30 mM Tris, 200 mM glycine, and 20% methanol. The transferred membranes were incubated at room temperature for 1 h with 5% bovine serum albumin (BSA) in 1× TBST (Tris-buffered saline containing 0.05% Tween 20) for blocking. Then, the membranes were incubated with the target primary antibody at 4 °C overnight, and following that, they were washed 3 times with 1× PBST or TBST for 10 min each. After this step, the membranes were treated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature, and then, the washing step was repeated another 3 times. The HRP was visualized using an Enhanced Chemiluminescence Femto kit (LPS solution, Daejeon, Republic of Korea) and a LAS-4000 Mini Lumino Image Analyzer (GE Healthcare Life Sciences, Chicago, IL, USA).
6
Electrophoretic Mobility Shift Assay for NigR
7
Western Blot Protein Analysis Protocol
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Cells were harvested to allow protein isolation using lysis buffer containing 1% Triton X-100 and protease inhibitor Complete (Roche, Mannheim, Germany). The cell lysate was then centrifuged at 15,500 × g for 15 min at 4°C, and the supernatant collected. Total protein was separated by SDS-15% polyacrylamide gel electrophoresis (PAGE) and transferred onto PVDF membranes (Millipore, Billerica, MA) using a Trans-Blot Semi-Dry Transfer Cell (Bio-Rad). The membrane was then blocked with 5% nonfat milk and probed with various antibodies as indicated. The immunoblot signal was detected using enhanced chemiluminescence reagent (PerkinElmer Life Sciences, Melbourne, Australia). The antibodies used in the study were anti-HBc antibody (Dako Cytomation, Glostrup, Denmark), anti-actin antibody (Sigma, St. Louis, MO), anti-P53 (Santa Cruz Biotechnology), anti-phospho-P53 (Santa Cruz Biotechnology), anti-P21(Calbiochem Biochemicals), anti-CEBP (GeneTex), anti-ERK (Merck Millipore), and anti-ERK-p (Cell Signaling Technology).
8
Chromatin Modifications and Transcription Analysis
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Western-blot analysis was performed on histones extracts prepared from 1-week-old seedlings as described previously (Xu et al., 2008 (link)). Protein were separated by 15% SDS-PAGE and transferred onto Immobilon-P membranes (Millipore) using a Trans-Blot semi-dry transfer cell (Bio-Rad). Intensity of individual bands was quantified using ImageJ densitometry software (NIH).
Chromatin immunoprecipitation (ChIP) assays were performed according to the previously described method (Liu et al., 2016 (link)). Antibodies used to precipitate chromatin were anti-H3 (05-499; Millipore), anti-trimethyl-H3K4 (07-473; Millipore), anti-trimethyl-H3K36 (ab9050; Abcam) and anti-total RNA polymerase II (RNAPII) CTD repeat antibody (ab817, Abcam), together with protein A magnetic beads (Magna-ChIP, Millipore). DNA was purified with the NucleoSpin Gel and PCR Clean-up kit (Macherey−Nagel, Düren, Germany) and analyzed by real-time PCR (LightCycler 480II; Roche in conjunction with the SYBR Green Master mix) using gene-specific primers listed inSupplementary Table S1 . Data were analyzed as described in Zhao et al., 2019 (link) for H3K4me3 and H3K36me3 and in Yang et al., 2016 (link) for RNAPII. A mock control was done using uncoupled magnetic beads (Supplementary Figure S5 ).
Chromatin immunoprecipitation (ChIP) assays were performed according to the previously described method (Liu et al., 2016 (link)). Antibodies used to precipitate chromatin were anti-H3 (05-499; Millipore), anti-trimethyl-H3K4 (07-473; Millipore), anti-trimethyl-H3K36 (ab9050; Abcam) and anti-total RNA polymerase II (RNAPII) CTD repeat antibody (ab817, Abcam), together with protein A magnetic beads (Magna-ChIP, Millipore). DNA was purified with the NucleoSpin Gel and PCR Clean-up kit (Macherey−Nagel, Düren, Germany) and analyzed by real-time PCR (LightCycler 480II; Roche in conjunction with the SYBR Green Master mix) using gene-specific primers listed in
9
Protein Extraction and Western Blot Analysis
10
Western Blot Protocol for Protein Analysis
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Spinal cord tissue was homogenized in lysis buffer with Tissue ruptor (Qiagen Instruments, Hombrechtikon, Switzerland). In vitro experiments were performed upon respective treatments, cells were scraped from the flask, cell pellets were lysed, cell lysates were centrifuged at 14,000 rpm in 4 °C for 30 min, and proteins were collected and stored at −20 °C. Protein concentrations were quantified (Pierce® BCA Protein Assay Kit, Thermo Scientific, Rockford, IL, USA) and normalized. Thirty micrograms of proteins were fractionated by denaturing gel electrophoresis (10% SDS-PAGE), transferred (Trans Blot, Semi dry Transfer cell, BioRad, Hercules, CA, USA) to a nitrocellulose membrane (GE Healthcare, AmershamTM Hybond ECL, Buckinghamshire, UK), and blocked with 5% BSA for 1 h. The membranes were incubated overnight at 4 °C with target-protein-specific primary antibodies, followed by 1 h incubation with respective secondary antibodies (Table 1 ). The immunoreactive bands of target proteins were detected with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA) using an ECL ChemoCam Imager (INTAS Science Imaging Instruments GmbH, Göttingen, Germany). GAPDH was used as a loading control. Protein band densities were analyzed by ImageJ 1.53b software (National Institute of Health, Bethesda, AR, USA).
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