The same LC and Fd expression cassettes as for the Fab extended by Vκ and VH outer domain encoding sequences, respectively, of trastuzumab spaced from the inner domains by ASTKGP encoding sequences were cloned to generate a HER2-targeting h38C2_Arg DVD-Fab (Nanna et al., 2017 (link)). Following expression in the Expi293F system described above, the culture supernatant was collected and purified by affinity chromatography with a 1-mL Protein A HP column (GE Healthcare) in conjunction with the ÄKTA FPLC instrument. The yield of DVD-Fab was ~18 mg/L as determined by the Pierce BCA Protein Assay Kit and its purity was confirmed by SDS-PAGE using a 10-well NuPAGE 4-12% Bis-Tris Protein Gel followed by staining with PageBlue Protein Staining Solution (all from Thermo Fisher). Using the same procedure, a DVD-Fab containing parental h38C2_Lys was cloned, expressed, and purified.
Nupage 4 12 bis tris protein gel
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany
The NuPAGE 4–12% Bis-Tris protein gels are pre-cast polyacrylamide gels designed for the separation and analysis of proteins. They provide a consistent and reliable platform for electrophoretic separation of proteins based on their molecular weight.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (80 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (49 mentions)
Ripa buffer, by Thermo Fisher Scientific (23 mentions)
Pvdf membrane, by Merck Group (22 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (18 mentions)
Nupage mops sds running buffer, by Thermo Fisher Scientific (17 mentions)
Dc protein assay, by Bio-Rad (17 mentions)
Iblot dry blotting system, by Thermo Fisher Scientific (16 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (16 mentions)
Simplyblue safestain, by Thermo Fisher Scientific (15 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (14 mentions)
Nupage sample reducing agent, by Thermo Fisher Scientific (14 mentions)
Odyssey blocking buffer, by LI COR (14 mentions)
Pvdf membrane, by Thermo Fisher Scientific (13 mentions)
Iblot 2 dry blotting system, by Thermo Fisher Scientific (13 mentions)
Nitrocellulose membrane, by Bio-Rad (13 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (12 mentions)
Bca protein assay, by Thermo Fisher Scientific (12 mentions)
Bca assay, by Thermo Fisher Scientific (11 mentions)
Protease inhibitor cocktail, by Roche (11 mentions)
372 protocols using nupage 4 12 bis tris protein gel
1
Generation and Purification of HER2-Targeting DVD-Fab
2
Immunoblotting of GFP-tagged Yeast Proteins
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Log-phase cultures of cells grown in YEP + 2% glucose were harvested and treated with 5% trichloroacetic acid (TCA) at 4°C overnight. TCA treated cell pellets were washed with acetone, air dried, and resuspended in lysis buffer (10 mM Tris, 1 mM EDTA, 2.75 mM DTT, pH = 8). Cells were lysed by bead-beating using a Multivortexer (max speed, 20 min) and glass beads at 4°C and followed by boiling in SDS PAGE protein loading buffer for 5 min. Lysates were clarified by centrifugation and were resolved on a 15-well NuPAGE 4–12% Bis-Tris protein gel (Thermo Fisher Scientific) prior to transfer onto nitrocellulose membranes. GFP-Mob1 and variants were detected using an anti-GFP antibody (Clontech, JL-8) at a 1:1000 dilution. Nud1-13myc and Cfi1/Net1-3myc were detected using an anti-Myc antibody (Abcam, 9E10) at a 1:500 dilution. Mob1-V5-TurboID was detected using an anti-V5 antibody (Invitrogen) at a 1:2000 dilution. Kar2 was detected using a rabbit anti-Kar2 antiserum at a 1:200,000 dilution. Secondary antibodies were used at a 1: 10,000 dilution. Blots were imaged using the ECL Plus system (GE Healthcare).
3
Western Blot Analysis of Protein Expression
4
Quantitative Western Blot Analysis
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After HP and ischemic stimulus, cells were washed with DPBS and lysed in lysis buffer containing RIPA lysis buffer (Cat. No.: 89901, Thermo Fisher Scientific), sodium metavanadate (Cat. No.: sc-251034, NaVO3, Santa Cruz Biotechnology), PMSF (Cat. No.: 36978, Thermo Fisher Scientific), and protease and phosphatase inhibitor cocktail (Cat. No.: 78440, Thermo Fisher Scientific). Protein concentration was determined by bicinchoninic acid (BCA) protein assay (Cat. No.: 23227, Thermo Scientific); denatured protein was resolved in a NuPAGE™ 4-12% Bis-Tris Protein Gel (Cat. No.: NP0323, Thermo Fisher Scientific) and transferred to a nitrocellulose membrane (Cat. No.: 77012, Thermo Fisher Scientific). Membranes were blocked and incubated overnight at 4°C with primary antibodies against AKT (Cat. No.: 9272, Cell Signaling Technology), phospho-AKT (Cat. No.: 4060, Cell Signaling Technology), and GAPDH (Cat. No.: sc-32233, Santa Cruz Biotechnology). Subsequently, membranes were incubated for 1 h with conjugated secondary antibodies (Cat. No.: 7074, Cell Signaling Technology) at room temperature and blots were imaged using a ChemiDoc MP+ imaging system (Bio-Rad), and further data analysis and gating were performed using ImageJ software.
5
Protein Extraction and Western Blot Analysis
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Hepatic tissues were treated with RIPA lysis buffer (P0013B, Beyotime, Haimen, China), and lysate was incubated on ice for 30 min. After centrifugation at 10, 000 × g for 5 min at 4°C, the protein concentration of each lysate was determined using a BCA Protein Assay Kit (P0010S, Beyotime). Total protein (20 μg) from each sample was boiled in SDS-PAGE loading buffer under reducing conditions, run on a NuPAGE™ 4-12% Bis-Tris Protein Gel (NP0322BOX, Thermo Fisher Scientific Inc.), and transferred to PVDF membranes (FFP24, Beyotime). The membranes were blocked with QuickBlock™ blocking buffer (P0222, Beyotime) in PBSTw, probed with primary antibodies, and followed by application of horseradish peroxidase-conjugated secondary antibodies [goat anti-mouse (BA1050, Wuhan Boster Biological Engineering Co., Ltd.) or goat anti-rabbit (BA1054, Wuhan Boster Biological Engineering Co., Ltd.)]. The signal was revealed by BeyoECL Plus Kit (P0018S, Wuhan Boster Biological Engineering Co., Ltd.) and imaged using an Odyssey FC imaging system (LI-COR Biosciences, Lincoln, NE, USA).
6
Quantification of FMRP in hiPSC and hESC Lysates
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hiPSC and hESC pellets were lysed using lysis buffer (50 mM Tris-HCL, pH 7.4, 2 mM EDTA, 0.1% SDS, 1% Triton-X 100, 0.5% Na-deoxycholate, 150 mM NaCl, protease and phosphatase inhibitor cocktails), sonicated (10 cycles of 30 s on/30 s off) and centrifuged at 13,000 rpm for 10 min. The supernatant was collected and protein estimation performed using BCA assay kit (Thermo Fisher Scientific). Twenty micrograms/sample protein was loaded on a precast NuPAGE 4–12% Bis-Tris Protein gel (Thermo Fisher Scientific) followed by protein transfer to a PVDF membrane (GE Healthcare, Life science, Chicago, IL). The blot was blocked in 1:1 TBST (0.2 M Trizma base, 0.15 M NaCl, 0.1% Tween-20) and Odyssey blocking buffer (Li-COR Bioscience, Lincoln, NE) for 1 h at room temperature. The membrane was incubated with primary antibodies FMRP (1:1000, AbCam, Cambridge, UK) and β-actin (1:5000, Sigma), in blocking buffer for overnight at 4 °C, washed with TBST and incubated in secondary antibodies with IRDye 680RD and IRDye 800CW (Li-COR Bioscience) respectively for 1 h at room temperature. Following further washing, the blot was dried and imaged using Li-COR Odyssey FC infrared system.
7
Western Blot Analysis Protocol
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For western blot analysis cells were washed with phosphate-buffered saline (PBS), harvested by using a cell lifter, and lysed in Radioimmunoprecipitation assay (RIPA) buffer with complete Mini EDTA-free protease inhibitor tablets (Roche) and phosphatase inhibitor cocktail PhosSTOP (Roche). The protein concentration was quantified using the BCA Assay (ThermoFischer Scientific) as described earlier [37 (link)]. 20 μg protein lysate were separated by SDS-gel electrophoresis using a NuPAGE™ 4–12% Bis-Tris protein gel and transferred to a nitrocellulose membrane using the iBlot Dry Blotting System (all ThermoFischer Scientific). As protein standards, 10 μl Spectra Multicolour Broad Range (ThermoFisher Scientific) and 1 μl MagicMark™ XP Western Protein Standard (ThermoFisher Scientific) were used. For detection, the membranes were incubated with WesternBright Sirius HRP substrate (Advansta). Except for the membranes displayed in S2C Fig , all signals were detected by a Microchemi chemiluminescence system (DNR Bio-Imaging Systems). S2C Fig had been digitalised by using an Odyssey CT (LI-COR). Antibodies used are listed in S2 Table . Densitometric analysis of experiments was made with the Image-Studio Lite 5.2 software (LI-COR). Uncropped western blot images are displayed in the supplementary files. Raw images files are displayed in S1 Raw images .
8
Protein Gel Electrophoresis and Viral RNA Analysis
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NuPAGE 4–12% Bis-Tris protein gel (Thermo Fisher Scientific, Waltham, MA, USA) was used for SDS-PAGE and Western blotting analyses. The chemiluminescence signals were detected using the LAS-3000 device (FUJIFILM, Tokyo, Japan). For detection of FLAG-tagged Met-IR and untagged full-length replication proteins of TMV, anti-DYKDDDDK antibody (clone 1E6; FUJIFILM Wako Pure Chemical, Osaka, Japan) and antisera against tomato mosaic virus replication proteins [8 (link)] were used, respectively. Luciferase activity was measured using the Renilla Luciferase Assay System (Promega) and the TD-20/20 luminometer (Promega). Radiolabeled RNA products were separated by 8 M urea–2.4% PAGE and detected using the Typhoon FLA 7000 scanner (GE Healthcare, Chicago, IL, USA). Band intensity of viral genomic RNA was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
9
Quantitative Analysis of Dnmt3b and Emerin
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Testis and mESCs were homogenized in lysis buffer (8 M Urea, 10% glycerol, 1% SDS, 5 mM DTT, 10 mM Tris pH 7.4). The lysates were sonicated for 10 cycles of 30 s with the Bioruptor (Pico; Diagenode, Ougrée, Belgium). After centrifugation, the supernatant was kept and protein concentrations were determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Bleiswijk, the Netherlands). Equal amounts of proteins were separated on a NuPAGE™ 4–12% Bis-Tris Protein Gel (Thermo Fisher Scientific, Bleiswijk, the Netherlands) and transferred to a Immobilon-FL PVDF membrane (Merck, Amsterdam, the Netherlands). The membrane was incubated in 4% skim milk/PBS overnight at 4 °C with the following antibodies: rabbit-anti-Dnmt3b antibody (ab2851; Abcam, Cambridge, UK; 1:600 dilution) and mouse-anti-emerin (NCL-emerin, clone 4G5; Novocastra; Leica Biosystems B.V., Amsterdam, the Netherlands, 1:375 dilution). The following day, the membrane was incubated with the secondary antibodies donkey-anti-rabbit-IRdye 800cw (1:5000 dilution) and donkey-anti-mouse-IRdye 680RD (1:5000 dilution) (Li-Cor, Bad Homburg, Germany) for 1 h at RT in 4% skim milk/PBS. The membrane was scanned with the Odyssey CLx imager (Li-Cor, Bad Homburg, Germany).
10
Whole Cell Lysis and Western Blot
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For whole cell lysis, cells were lysed by direct addition of 2% SDS, 100 mM Tris/HCl pH 7.5 and sonicated with a sonicator bath (Bioruptor Pico—Rm 343) for 15 cycles. Sample concentration was adjusted with a Pierce BCA Protein Assay Kit (Thermo) before the addition of NuPAGE LDS Sample Buffer (Thermo) with reducing agent and boiling at 95°C for 10 min. After separation on a NuPage 4–12% Bis/Tris protein gel (Thermo), proteins were transferred to an Immobilon‐P membrane (Millipore) using a standard wet transfer device. Primary antibodies were diluted in 5%BSA, PBS and incubated on the membranes at 4°C overnight followed by incubation with anti‐mouse or rabbit HRP‐conjugated secondary antibodies at RT for 1 h. Membranes were then probed with Pierce ECL Plus HRP‐detection reagent followed by imaging on an Amersham Imager 600. Immunoblots (IBs) were quantified using ImageJ.
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