Nucleosome binding tests were performed using purified HeLa mononucleosomes (Epicypher, 16–0002) resolved by 15% SDS-PAGE, transferred, and probed similar to a Western blot with modifications. For each lane, 1 μg of nucleosomes was run and after transfer, lanes were separated for individual incubation with the indicated reader domain or antibody. After blocking with 5% non-fat milk in 1 X TBST (10 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 7.6) for 15 minutes, a single lane was incubated with the indicated concentration of chromodomain in blocking buffer (or blocking buffer alone for the blots used for the commercial antibody) for one hour at room temperature with shaking. The strip blots were then washed three times for five minutes with 1 X TBST and subsequently incubated with 1:1000 anti-MBP antibody (ThermoFisher, PA1–989) or the indicated concentration of anti-H3K9me3 antibody (Active Motif, 39161) for one hour at room temperature with shaking. The blots were again washed three times for five minutes with 1 X TBST, after which they were incubated with anti-Rabbit-HRP at 1:20,000 (GE, NA934V) in blocking buffer for one hour, with shaking, at room temperature. Last, the blots were washed three times for five minutes and then imaged after incubation with a chemiluminescent substrate according to the manufacturer (GE, RPN2232).
Rpn2232
Manufactured by GE Healthcare
Sourced in United States, United Kingdom
The RPN2232 is a laboratory instrument designed for sample preparation and analysis. It features automated handling and processing capabilities to support various laboratory workflows. The core function of the RPN2232 is to provide consistent and reliable performance in sample preparation tasks, without further interpretation of its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Roche (4 mentions)
Na934v, by GE Healthcare (3 mentions)
Rpn2106, by GE Healthcare (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
P8340, by Merck Group (2 mentions)
Quantity analysis software, by Bio-Rad (2 mentions)
Mab4864, by R&D Systems (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Dc protein assay, by Bio-Rad (2 mentions)
Na931v, by Santa Cruz Biotechnology (2 mentions)
Na934v, by Santa Cruz Biotechnology (2 mentions)
Anti mouse and anti rabbit, by GE Healthcare (2 mentions)
Anti goat, by Santa Cruz Biotechnology (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Sds page gel, by Bio-Rad (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
48 protocols using rpn2232
1
Nucleosome Binding Assay Protocol
2
Western Blot Analysis of LAMP3, E-cadherin, and β-actin
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The protein expression levels of LAMP3, E-cadherin, and β-actin were analyzed by western blot assay. Following treatments, cells were harvested in radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) supplemented with protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA). Equal amount of protein (~20 µg) was resolved in 12% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. Following blocking with 5% non-fat dry milk in TBS/0.1% Tween-20 (TBST) buffer, membranes were incubated with primary antibodies at 4°C overnight. Following three washes with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. Following washing, the membranes were developed using chemiluminescence substrate (RPN2232; GE Healthcare Life Sciences, Little Chalfont, UK) and imaged. The antibodies used were: Mouse anti-β-actin (ab8227; 1:5,000; Abcam), anti-mouse HRP-conjugated IgG (A9044; 1:5,000; Sigma-Aldrich; Merck KGaA), anti-rabbit HRP-conjugated IgG (A0545; 1:5,000; Sigma-Aldrich; Merck KGaA), anti-E-Cadherin (3195; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-LAMP3 (ab83659; 1:1,000; Abcam).
3
Western Blot Analysis of Recombinant Proteins
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To verify the induction of recombinant proteins, 2 ml of bacterial culture were harvested before and 6 h after IPTG induction; cells were pelleted by centrifugation at 4000 rpm for 5 min. The pellet was washed and resuspended with 1 ml 1x PBS buffer (1 mM KH2PO4, 10 mM NaHPO4, 137 mM NaCl, 2.7 mM KCl) and cells lysed by sonication. Cell debris was pelleted by centrifugation (14,000 rpm, 30 min, 4 °C) and the supernatant was collected. Ten microliter of 4x Laemmli sample buffer (Biorad) was added to 30 μl of supernatant and denatured by boiling at 95 °C for 5 min. Samples were then loaded on a 12% (w/v) SDS/PAGE gel and, after gel run, transferred to a PVDF membrane by wet electroblotting. For detection of recombinant proteins, the PVDF membrane was blocked in 1x TBS-T with 5% skimmed milk for 2 h at room temperature. After blocking, the membrane was incubated with anti-6x His antibody horseradish peroxidase (HRP) conjugate (Sigma A7058-1VL, 1:10,000 dilution in 1x TBS-T with 5% skimmed milk) for 2 h at room temperature and washed for 10 min, four times, in 1x TBS-T. Signals were detected by an enhanced chemiluminescent (ECL) detection system (GE Healthcare RPN2232). ECL signals were captured by a charge-coupled device (CCD) camera (Azure c600) (Additional file 2 : Figure S1).
4
Western Blotting Analysis of EMT Markers
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All the Western blotting analyses were performed as described previously (Zhao et al., 2015 (link); Chen et al., 2017a (link),c (link); Wang et al., 2017a (link)). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and transferred to polyvinylidene difluoride (PVDF) membranes (10600023, AmershamTM HybondTM, GE Healthcare, United States) by electroblotting. After blocking in Tween-20 buffer and non-fat milk blocking buffer, the membranes were incubated at 4°C over-night with primary antibodies against proteins including E-cadherin (1:500, Abcam, United States), FSP1 (1:1000, Abcam, United States), vimentin (1:200, Abcam, United States), and collagen III (1:5000, Abcam, United States). Membranes were washed and incubated with appropriate horseradish peroxidase-conjugated goat anti-mouse (1:5000, Abbkine, United States) and goat anti-rabbit (1:5000, Abcam, United States) secondary antibodies for 1 h. Blots were detected by enhanced chemiluminescence (RPN2232, GE Healthcare, United States) and band density was quantified by Image J 1.48v software. GAPDH served as the internal control.
5
Western Blot Analysis of Francisella Proteins
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Unless stated otherwise, lysates from bacteria grown on modified GC agar were prepared in Laemmli sample buffer and boiled prior to separation on 12–15% sodium dodecyl sulfate (SDS)-polyacrylamide gels (161–0175, Bio-Rad). Proteins were transferred onto nitrocellulose membranes (170–4158, Bio-Rad) using a semidry blotter (Bio-Rad laboratories, Hercules, CA). Membranes were probed with mouse monoclonal antibodies recognizing IglC or PdpB at 1/2,000 and 1/1,000 dilutions respectively (NR-3196 and NR-3198, BEI Resources), rabbit polyclonal antibodies recognizing VgrG at 1/1,000 dilution (Inbiolabs, Tallinn, Estonia), mouse monoclonal antibodies recognizing Tul4 (1/3,000 dilution) (kind gift from Dr J. Stulik, Czech Republic), rat polyclonal antibodies recognizing IglE 34 (link) (1/2,000 dilution). GSK-tagged variants of IglE were detected using rabbit anti-GSK antibodies (1/1,000 dilution) (9336S, Cell Signaling Technology). The secondary horseradish peroxidase (HRP)-conjugated antibodies used were: goat anti-mouse and goat anti-rat (S-2005 and S-2032, Santa Cruz Biotechnology) at 1/2,000 and 1/5,000 dilutions respectively, and donkey anti-rabbit (NA934V, GE Healthcare) at 1/13,300 dilution. For detection, the Enhanced Chemiluminescence system (ECL; RPN 2232, GE Healthcare) was used.
6
Western Blot Analysis of Protein Expression
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The cells were lysed in Cell Lysis Buffer (#9803, Cell Signaling Technology) containing PMSF (022-15371, Wako) and protease inhibitors (P8340, Sigma). The cell lysates were suspended in Lane Marker Reducing Sample Buffer (Cat#39000, Thermo Scientific) for protein concentrations of 1–2 mg/mL, and then boiled at 95 °C for 5 min. Five–ten-micrograms of protein were electrophoresed in 4–20% SDS-PAGE gels (Bio-Rad, CA) and transferred onto a PVDF membrane (Millipore, MA). After blocking with 4% bovine serum albumin (10-735-108-001, Roche), the membrane was incubated with the primary antibodies (1:1000 dilution) followed by incubation with horse radish peroxidase-conjugated secondary antibodies against anti-mouse IgG (#7076, Cell Signaling Technology) or anti-rabbit IgG (#7074, Cell Signaling Technology) (1:5000 dilution). The immunoblotted proteins were visualized by luminol-based enhanced chemiluminescence (RPN2232, GE healthcare), and the luminescent images were captured by ImageQuant LAS 4010 (GE healthcare). We processed images of electrophoretic gels and blots according to the digital image and integrity policies of Scientific Reports.
7
Western Blot Analysis of SERCA2 and β-Actin
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Equal amounts of protein were separated on 10% sodium dodecyl sulfate‐polyacrylamide gels for electrophoresis and then transferred to the nitrocellulose membrane. Primary SERCA2 (1:1000 dilution) and β‐actin (1:10000 dilution) antibodies diluted in TBS containing 5% nonfat dried milk and 0.05% tween‐20 were used to determine the corresponding protein expression. Then secondary antibodies (HRP‐conjugated goat anti‐rabbit IgG or goat anti‐mouse IgG) were tagged with primary β‐actin (1:20000 dilution) and SERCA2 (1:2000 dilution) antibodies. The immunoreactive bands were detected with a chemiluminescent substrate (RPN2232, GE Healthcare). The immunoreactive signals were quantified by quantity analysis software (Bio‐Rad).
8
Quantification of m6A RNA Methylation
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mRNA was purified by a Dynabeads mRNA purification kit (Ambion, 61006) and quantified using ultraviolet spectrophotometry. The m6A dot blot was performed on Bio-Dot Apparatus (Bio-Rad, no. 170-6545). In brief, the primary rabbit anti-m6A antibody (Synaptic Systems, 202003) was applied to the charged nylon-based membrane containing mRNA samples. HRP-conjugated goat anti-rabbit IgG (DakoCytomation, p0448) was added to the blots and then developed with enhanced chemiluminescence (GE Healthcare, RPN2232). The signal density of the dot-blot experiment is quantified with Gel-Pro analyzer software (Media Cybernetics) in all experiments.
9
Protein Expression Analysis of ALI-CO Slices
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Cell lysates for protein samples were obtained from ALI-CO slices at 150, 200, 220 and 240 DIV using RIPA lysis buffer (Sigma-Aldrich, R0278) plus protein and phosphatase inhibitors (Thermo Fisher Scientific, 31462, A32957), and standard immunoblotting protocols were used. Briefly, 18 μg of protein was resolved by SDS–PAGE then transferred onto polyvinylidenedifluoride membranes before overnight incubation with primary antibodies (Supplementary Table 2 ), except for the directly conjugated β-actin antibody, for which 1-h incubation was applied. Species-specific horseradish-peroxidase-conjugated secondary antibodies were applied for 1 h at room temperature at 1:10,000 dilution (Supplementary Table 2 ) before signal detection with the enhanced chemiluminescence system (GE Healthcare, RPN2232). Standard quantitative WB analysis was performed in ImageJ, and band density levels were expressed as fold-changes to controls after normalization to β-actin and controls within the same blot.
10
Western Blot Protein Quantification
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