ESCs or EBs were lysed with RIPA buffer (Pierce) in the presence of EDTA free protease inhibitor cocktail (Sigma). Histone extraction on EBs was performed using a histone extraction kit (Abcam) as described previously (Khan et al., 2015 (link)). Protein concentration was determined using Pierce BCA protein assay kit (ThermoFisher Scientific). 1.0 μg of histone extracts, or 10 μg of whole cell lysate was resolved on 4%–20% precast gel (Bio-Rad) was transferred to 0.45 μm PVDF membrane (Millipore). For immunoprecipitation, day 2 WT and Wdr5KO EBs were harvested and lysed in RIPA buffer with EDTA free protease inhibitor cocktail (Sigma). 1 mg sonicated cell lysates were subjected to immunoprecipitation using WDR5 antibody (Bethyl). The following primary antibodies were used for probing: anti-HA (1:10,000, Abcam, ab9110), anti-WDR5 (1:5000, R&D), anti-WDR5 (1:5,000, Bethyl), anti-Flag (1:2,000, Sigma), anti-H3 (1:10,000, Abcam), anti-H3K4Me1 (1:5,000, Millipore), anti-H3K4Me2 (1:10,000, Millipore), anti-H3K4Me3 (1:10,000, Abcam), anti-P53 (1:5,000, Cell Signaling), anti-Tubulin (1:10,000, Cell signaling), anti-β-Actin (1:10,000, Cell signaling), anti-Phospho-p53 (Ser15) Antibody (1:5,000, Cell Signaling), anti-Phospho-p53 (Ser392) Antibody (1:5,000, Abcam), anti-Acetyl-p53 (K305) Antibody (1:5,000, Abcam), anti-Actyl-p53 (K379) Antibody (1:5,000, Cell Signaling).
Edta free protease inhibitor cocktail
Manufactured by Merck Group
Sourced in United States, Germany
EDTA-free protease inhibitor cocktail is a laboratory reagent designed to inhibit a broad range of protease enzymes without the presence of EDTA. It is used to prevent protein degradation during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Merck Group (6 mentions)
Phosstop, by Merck Group (4 mentions)
Glutathione sepharose 4b beads, by GE Healthcare (4 mentions)
Dc protein assay kit, by Bio-Rad (3 mentions)
Odyssey clx, by LI COR (3 mentions)
Benzonase, by Merck Group (3 mentions)
Phosstop phosphatase inhibitor cocktail, by Merck Group (3 mentions)
Irdye 800cw goat anti mouse, by LI COR (3 mentions)
Irdye 680rd goat anti rabbit, by LI COR (3 mentions)
Phosphatase inhibitor cocktail 3, by Merck Group (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Complete mini, by Merck Group (2 mentions)
Pierce universal nuclease, by Thermo Fisher Scientific (2 mentions)
Clx imaging device, by LI COR (2 mentions)
Ab52866, by Abcam (2 mentions)
Ab97046, by Abcam (2 mentions)
Chemicoc mp imaging system, by Bio-Rad (2 mentions)
Luminata crescendo western hrp substrate, by Bio-Rad (2 mentions)
Laemmli sample buffer, by Bio-Rad (2 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (2 mentions)
104 protocols using edta free protease inhibitor cocktail
1
Histone Extraction and Immunoprecipitation from Embryonic Stem Cells
2
Histone Extraction and Immunoprecipitation from Embryonic Stem Cells
3
Protein Extraction and Western Blot Analysis
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For preparation of protein extracts, HBEpC were lysed using RIPA buffer (150 mM NaCl, 50 mM Tris/HCl pH 8.0, 1% (v/v) Triton X-100, 0.5% (v/v) sodium deoxycholate, 0.3% (v/v) SDS, 5 mM NaF, 1 mM Na3VO4, 1x cOmplete Mini, EDTA-free protease inhibitor cocktail (Merck), 2 mM MgCl2 and 1 U/mL PierceTM Universal Nuclease (Thermo Scientific). Protein concentrations were determined using DC Protein Assay Kit (Bio-Rad, Hercules, CA, USA) samples were separated by SDS-PAGE and were blotted onto AmershamTM ProtranTM Premium 0.45 µm nitrocellulose membrane (Cytiva, Marlborough, MA, USA) for 1.5 h using const. 120 V. Membranes were blocked for 1 h in 5% (w/v) BSA in TBS-t (150 mM NaCl, 20 mM Tris, 0.1% (v/v) Tween-20) and incubated with SARS-CoV-2 (2019-nCoV) Spike S1 Antibody (1:1000, #40150-R007, Sino Biological, Beijing, China), SARS-CoV/SARS-CoV-2 Nucleocapsid antibody (1:1000, #40143-MM05, Sino Biological, Beijing, China) and β-actin antibody (1:1000, #4967, Cell Signaling Technology, Leiden, The Netherlands) overnight at 4 °C in blocking buffer. Secondary antibodies IRDye® 800CW goat anti-rabbit IgG (H + L) and IRDye® 680RD goat anti-mouse IgG (H + L) (LI-COR Biosciences GmbH, Bad Homburg, Germany) were diluted 1:40.000 in blocking buffer and incubated for 1 h at room temperature. Blots were imaged using CLx imaging device (LI-COR Biosciences GmbH, Bad Homburg, Germany).
4
Quantifying PDH Protein in iNs
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PDH protein was quantified in lysed pellets of FACS-purified iNs using the ProteinSimple Jess (Biotechne) and the 12-230 kDa Jess Separation Module in the NIR channel. Pellets were lysed in RIPA Lysis and extraction Buffer (ThermoFisher) containing cOmplete EDTA-free Protease Inhibitor Cocktail (Merck, 11873580001) and PhosSTOP (Merck, 4906845001). Anti-PDH antibody (Santa Cruz, sc-377092, 1:50) was incubated for 60 minutes, followed by standard default run settings provided by ProteinSimple. Data analysis was performed using Compass software.
5
Recombinant G6PD Protein Expression
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Genes for wild-type G6PD and G6PDViangchan were transformed into Escherichia coli BL21 (DE3) competent cells for protein expression. At log phase, OD600nm 0.8, the culture was induced with 0.1 mM and 1 mM isopropyl β-d -1-thiogalactopyranoside (IPTG) and grown at 16°C, 25°C, and 37°C for 3, 5, 12, and 16 h. Cell lysate was centrifuged at 4,000 rpm for 10 min using an Eppendorf Benchtop 5424 Microcentrifuge with an FA-45-11 rotor. The pellets were resuspended with lysis buffer (0.1 M Tris-HCl, pH 7.6, 3 mM MgCl2, 0.5 mM EDTA free protease inhibitor cocktail (Merck), and 0.1% β-mercaptoethanol). The cells were sonicated on ice with 10 short pulses (10 s) followed by pauses (30 s). The suspension was centrifuged at 15,000 rpm using an Eppendorf Benchtop 5424 Microcentrifuge with an FA-45-11 rotor for 15 min at 4°C. Supernatants were collected as soluble protein. Protein concentration was determined using a Pierce bicinchoninic acid protein assay kit (Thermo Fisher Scientific) with bovine serum albumin as the protein standard, before sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. All G6PD proteins expressed were C-terminally tagged with histidine (His-Tag).
6
Recombinant SPANXA Protein Purification
7
Heterologous Expression of GLUT4 and AQP7
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Rat GLUT4 with N‐terminal poly‐histidine tag and human AQP7 (Aquaporin 7, as a control in far dot western blot) with C‐terminal His‐tag were cloned into the double‐deletion strain Pichia pastoris GS115 aqy1Δ::HIS4 agp1Δ::NatMX with no endogenous aquaporins and aquaglyceroporins present [21 (link)]. The yeast cells with GLUT4 and AQP7 construct were cultured as previously described [22 (link)]. Harvested cells were lysed with high‐pressure homogenization apparatus (Biox AB, Onsala, Sweden), subsequently, membranes were isolated by ultra‐centrifugation at 145 000 x g for 90 min at 4 °C and solubilized in 20 mM sodium phosphate pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM DTT, EDTA‐free protease inhibitor cocktail (Merck, Darmstadt, Germany) and 1% decyl β‐D‐maltopyranoside (DM, Anatrace, Maumee, OH, USA) for 1 h at 4 °C. Homogenate was clarified by centrifugation at 145 000 x g for 30 min, and subjected to western blot analysis. In brief, the PVDF membrane was blocked in 2% fish gelatin before incubating with GLUT4 antibody [18 (link)] and corresponding HRP conjugated secondary antibody (Invitrogen, Waltham, MA, USA) to identify GLUT4 protein in the lysate. Finally, the signal was recorded with Odyssey Fc gel‐scanning system (LI‐COR, Lincoln, NE, USA).
8
S. suis Infection Induces Apoptosis
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PAM-Tang cells were cultured in 12-well plates at a density of 1 × 106 cells/well in RPMI 1640 supplemented with 10% FBS. Thirty minutes prior to infection, the medium was replaced with RPMI 1640 with 3% FBS. Cells were then infected with S. suis 700794 or S. suis W7119 (MOI = 1) for 0, 9, or 16 h, and subsequently incubated on ice with cell lysis buffer containing PMSF (Solarbio, Beijing, China) and EDTA-free protease inhibitor cocktail (Roche, Merck KGaA, Mannheim, Germany) for 5 min. Protein concentrations were determined using Bicinchoninic Acid Protein Assay kit (Beyotime, Beijing, China). Equal amounts of protein were separated on a 12% SDS-PAGE gel and transferred to polyvinylidene fluoride (PVDF) membranes (MerckMillipore, Darmstadt, Germany) for 1 h. After blocking with 5% dry milk dissolved in PBS at 4 °C overnight, membranes were incubated for 1 h at room temperature with different antibodies: anti-caspase-3, anti-apoptosis-inducing factor (AIF) (1:1000, ABclonal), and anti-β-actin (1:200,000; ABclonal). After washing, the membranes were incubated with the appropriate secondary antibodies for 1 h, and visualized on the near-infrared fluorescence scanning imaging system (Odyssey CLx, Licor, Lincoln, NE, USA) to detect the target bands.
9
Quantification of Poly(GA) and Poly(GP) Expression
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Cells were lysed in RIPA buffer (Sigma-Aldrich) supplemented with 2× complete Mini, EDTA free protease inhibitor cocktail (Merck), and 2% SDS (ThermoFisher) and homogenized by sonication. After centrifugation at 17,000 × g at 16 °C for 20 min, the protein content of the supernatants was determined as described in the Supplementary Methods. The samples were adjusted to the same concentration (0.6–1.0 mg/mL) and MSD immunoassay was performed in singleplex using 96-well SECTOR plates (MSD) to quantify Poly(GA)/(GP) expression, as described previously20 (link). In brief, plates were coated with unlabeled anti-poly(GP) or anti-poly(GA) antibodies. After blocking, samples were loaded at 45 µg of protein per well for the poly(GP) immunoassay and at 27 µg of protein for poly(GA). Biotinylated anti-poly(GP) and anti-poly(GA) antibodies were used as detectors, followed by sulfo-tagged streptavidin (Meso Scale Discovery, R32AD). Plates were read with the MSD reading buffer (Meso Scale Discovery, R92TC) using the MSD Sector Imager 2400. Signals correspond to intensity of emitted light upon electrochemical stimulation of the assay plate. Prior to analysis, the average reading from a calibrator containing no peptide was subtracted from each reading. Negative signals were set to ‘0’.
10
Purification of Recombinant Proteins
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Cells were grown in 300 mL of LB media with 50 μg mL−1 kanamycin at 37 °C and 250 rpm until OD600 reached ~0.6, and then the culture was induced with 1 mM IPTG and allowed to express at 18 °C for 20 h. Proteins were purified on a nickel column with a Bio-Rad BioLogic LP FPLC (Wash buffer: 50 mM sodium phosphate, pH 7.2, 300 mM NaCl, 20 mM imidazole, and EDTA-free protease inhibitor cocktail (Roche); Elution buffer: 50 mM sodium phosphate, pH 7.2, 300 mM NaCl, 200 mM imidazole, and EDTA-free protease inhibitor cocktail), concentrated with a 10 kDa MWCO filter (EMD Millipore), and stored as single-use aliquots in module storage buffer (100 mM sodium phosphate, pH 7.4, 1 mM EDTA, 1 mM tris(2-carboxyethyl)phosphine (TCEP), 20% v/v glycerol, 0.1 μL Benzonase (NEB), and EDTA-free protease inhibitor cocktail) at −80 °C.
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