The expression of keratinocyte markers before and after differentiation of ASCs into adipocytes was assessed by western blotting. ASCs were lysed in IGEPAL Nonidet P-40 in the presence of Halt Protease and Phosphatase Inhibitor Cocktail (both from Sigma-Aldrich). Proteins were separated by 10% Tris-glycine SDS-PAGE (Bio-Rad, Hercules, CA, USA) under denaturing conditions and transferred to a nitrocellulose membrane. After blocking with 3% bovine serum albumin in Tris-buffered saline, the membrane was incubated with primary antibodies against p63 (Cell Signaling Technology) and DSG3 (Santa Cruz Biotechnology) overnight at 4°C. The blot was proved for β-actin using a monoclonal antibody (1:2000; BioLegend, San Diego, CA, USA) as a loading control. The membrane was then washed, incubated with anti-mouse or -rabbit peroxidase-conjugated secondary antibody (1:1000; Santa Cruz Biotechnology and Cell Signaling Technology, respectively) at room temperature for 45 min, and developed with Luminate Forte western horseradish peroxidase substrate (Merck Millipore, Billerica, MA, USA).
Tris glycine sds page
Manufactured by Bio-Rad
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Tris-Glycine SDS-PAGE is a type of gel electrophoresis system used for the separation and analysis of proteins based on their molecular weight. It utilizes a discontinuous buffer system with Tris-glycine as the running buffer and sodium dodecyl sulfate (SDS) as the denaturing agent.
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Lab products found in correlation
Laemmli sample buffer, by Bio-Rad (3 mentions)
Halt protease, by Merck Group (2 mentions)
Phosphatase inhibitor cocktail, by Merck Group (2 mentions)
Igepal nonidet p 40, by Merck Group (2 mentions)
A10043, by LI COR (2 mentions)
Trizol kit, by Thermo Fisher Scientific (2 mentions)
Sigma sab2502119, by Merck Group (2 mentions)
A21088, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Dsg 3, by Santa Cruz Biotechnology (1 mentions)
Luminata forte western horseradish peroxidase substrate, by Merck Group (1 mentions)
Hmb45, by Santa Cruz Biotechnology (1 mentions)
Instantblue, by Abcam (1 mentions)
Pageblue, by Thermo Fisher Scientific (1 mentions)
Superdex 200 pc 3.2 column, by GE Healthcare (1 mentions)
Intercept blocking buffer, by LI COR (1 mentions)
Empiria studio software, by LI COR (1 mentions)
Irdye 800cw, by LI COR (1 mentions)
15 protocols using tris glycine sds page
1
Keratinocyte Markers in ASC Adipogenesis
2
Melanocytic Marker Expression in ADSCs
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The expression of melanocytic markers in undifferentiated and differentiated ADSCs was assessed by western blotting. NHEM were used as a positive control.
Cells were lysed in IGEPAL Nonidet P-40 in the presence of Halt Protease and Phosphatase Inhibitor Cocktail (both from Sigma-Aldrich). Proteins were separated by 10% Tris-glycine SDS-PAGE (Bio-Rad, Hercules, CA, USA) under denaturing conditions and transferred to a nitrocellulose membrane. After blocking with 3% bovine serum albumin in Tris-buffered saline, the membrane was incubated with primary antibodies against HMB45 (1:200; Santa Cruz Biotechnology) and MITF (1 μg/ml; Abcam Biotechnology) overnight at 4°C. The blot was probed for β-actin using a monoclonal antibody (1:2000; BioLegend, San Diego, CA, USA) as a loading control. The membrane was then washed, incubated with an anti-mouse peroxidase-conjugated secondary antibody (1:1000; Santa Cruz Biotechnology and Cell Signaling Technology) and anti-rabbit antibody (1:3000; Cell Signaling Technology) at room temperature for 45 min, and developed with Luminata Forte Western horseradish peroxidase substrate (Merck Millipore, Billerica, MA, USA).
Cells were lysed in IGEPAL Nonidet P-40 in the presence of Halt Protease and Phosphatase Inhibitor Cocktail (both from Sigma-Aldrich). Proteins were separated by 10% Tris-glycine SDS-PAGE (Bio-Rad, Hercules, CA, USA) under denaturing conditions and transferred to a nitrocellulose membrane. After blocking with 3% bovine serum albumin in Tris-buffered saline, the membrane was incubated with primary antibodies against HMB45 (1:200; Santa Cruz Biotechnology) and MITF (1 μg/ml; Abcam Biotechnology) overnight at 4°C. The blot was probed for β-actin using a monoclonal antibody (1:2000; BioLegend, San Diego, CA, USA) as a loading control. The membrane was then washed, incubated with an anti-mouse peroxidase-conjugated secondary antibody (1:1000; Santa Cruz Biotechnology and Cell Signaling Technology) and anti-rabbit antibody (1:3000; Cell Signaling Technology) at room temperature for 45 min, and developed with Luminata Forte Western horseradish peroxidase substrate (Merck Millipore, Billerica, MA, USA).
3
Inhibition Assay for Serine Protease GlpG
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For inhibition assays, the purified MBP-TatAtmd-Trx fusion protein encompassing amino acids 1–50 of P. stuartii TatA (Strisovsky et al, 2009 (link)) was used as substrate. Purified full-length GlpG (5.4 μM) was preincubated with peptidyl-chloromethylketone inhibitors at different concentrations (50–700 μM) for 3 h at 37°C in reaction buffer containing 50 mM Tris (pH 7.4), 100 mM NaCl, 25 mM EDTA, 10 % (v/v) glycerol and 0.05 % (v/v) DDM. The cleavage reaction was started by adding substrate in fivefold molar excess over the enzyme, and let proceed for 30 min at 37°C, after which it was stopped by the addition of SDS-PAGE sample buffer and transfer on ice. Reaction products were resolved by 4–20% Tris-Glycine SDS-PAGE (Bio-Rad) and Coomassie stained (Instant Blue, Expedeon, UK). Substrate conversion was quantified densitometrically from the scanned stained gels using the ImageQuant 8.0 software (GE Healthcare).
4
Probing MLKL-Dependent Cell Death
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MLKL-/- HT29 cells were seeded into 48-well plates at 60,000 cells/well and left to settle for 6 h. Cells were then induced overnight with doxycycline (100 ng/mL) to stimulate MLKL expression. Following doxycycline pretreatment, cells were stimulated with TNF (100 ng/mL), Smac mimetic Compound A (500 nM), and Pan-caspase inhibitor IDN-6556 (5 µM) for 4 h. Cells were harvested in 2× SDS Laemmli’s lysis buffer, boiled at 100 °C for 10–15 min, and then resolved by 4–15% Tris-Glycine SDS-PAGE (Bio-Rad). Proteins were transferred to PVDF membrane and probed with the antibodies indicated.
5
NisB-NisC-Prenisin Co-elution Analysis
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The co-elution studies were performed on a Äkta Micro system using a Superdex 200 PC 3.2 column (GE Healthcare) equilibrated with 50 mM HEPES-NaOH, pH 7.5, 500 mM NaCl with a flow rate of 0.05 mL/min.
A 50 μL reaction mixture consisting of 20 μM NisB, 160 μM NisC and 200 μM prenisin peptide variant was incubated for 1 h at 25 °C and subsequently applied to SEC analysis. Elution was observed at 280 nm. After co-elution, the corresponding fractions were analyzed by a 4–20% gradient Tris-Glycine SDS-PAGE (Biorad) gel stained with Page-Blue (Thermo Fisher).
A 50 μL reaction mixture consisting of 20 μM NisB, 160 μM NisC and 200 μM prenisin peptide variant was incubated for 1 h at 25 °C and subsequently applied to SEC analysis. Elution was observed at 280 nm. After co-elution, the corresponding fractions were analyzed by a 4–20% gradient Tris-Glycine SDS-PAGE (Biorad) gel stained with Page-Blue (Thermo Fisher).
6
Quantitative Western Blot Analysis of Complement Proteins
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Proteins were separated by 4–15% Tris–Glycine SDS-PAGE (BioRad, 456-1086) under non-reducing conditions. Gels included standard lanes of C3a-desArg (32–1.25 ng/lane) and Ba (20–2.5 ng/lane) proteins for quantification and a pre-stained protein ladder (LI-COR, 928-6000) for approximating size. Gels were transferred using a semi-dry method (BioRad Trans-Blot, 170-3940) onto low-fluorescence PVDF membranes (IPFL 10100). Remaining steps followed protocols recommended by LI-COR using TBS-based Intercept Blocking Buffer (LI-COR, 927-00001) for membrane blocking and antibody dilution. Primary antibodies were diluted in Intercept buffer containing 0.2% Tween-20 and diluent for fluorescent secondary antibodies contained 0.2% Tween-20 plus 0.01% SDS. Ba protein was detected using goat anti-human FB (4.3 µg/ml) plus donkey anti-goat IRDye-680RD (1:20,000, LI-COR, 926-68074). C3a protein was detected using rabbit anti-human C3a (6.9 µg/ml) plus donkey anti-rabbit IRDye-800CW (1:20,000, LI-COR, 926-32213). Dried membranes were scanned on a LI-COR Odyssey DLx Imager (9142-01P), intensities were measured at 700 and 800 nm, and data were analyzed using Empiria Studio software (LI-COR, 2000-000).
7
Western Blot Analysis of Retinal Vascular Proteins
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Western blot analysis was performed as described in our previous study of retinal arterioles with slight modification.26 (link),46 (link) Retinal venules (sample pooled from both eyes per pig) and neural retina tissue were isolated and sonicated in lysis buffer. The protein content of each lysate was determined with the BCA protein assay kit (Pierce, Rockford, IL, USA). Equal amounts of protein (2.5 μg for ET-1 receptors and 5 μg for ROCK isoforms and p38) were separated by Tris-glycine SDS-PAGE (4%–15% Tris-HCl Ready Gels; Bio-Rad, Hercules, CA, USA), transferred onto a nitrocellulose membrane, and incubated with rabbit anti-ETAR or anti-ETBR polyclonal antibody (1:250 dilution; Catalog nos. sc-33535 and sc-33537; Santa Cruz Biotechnology, Santa Cruz, CA, USA),26 (link) or mouse anti-ROCK1 or anti-ROCK2 monoclonal antibody (1:250 dilution; Catalog nos. sc-17794 and sc-398519; Santa Cruz Biotechnology). Membranes were stripped and reprobed with rabbit anti-p38 antibody (1:1000; Catalog no. sc-535; Santa Cruz Biotechnology), which we previously have shown is highly expressed in neural retina tissue.26 (link) After incubation with an appropriate secondary antibody (anti-rabbit or anti-mouse IgG, 1:1000; Catalog nos. 7074S and 7076S; Cell Signaling Technology, Danvers, MA, USA), the membranes were washed and developed by enhanced chemiluminescence (Pierce).
8
Quantifying Protein Expression in Ischemic Muscle and BMDMs
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Proteins from ischemic muscle tissue (100–150 mg) were extracted using Trizol kit (Fisher 15596026). Proteins from BMDMs (1.5million/mL) were collected by using Laemmli sample buffer (Bio-Rad, cat.1610737) containing 2-βME; then boiled at 95°C and aliquoted. Aliquots from tissue lysates or cell lysates were separated by Tris/Glycine/SDS–PAGE (Bio-Rad 1610732) and blotted into PVDF membranes. Membranes were blocked in fluorescent blocking buffer (Rockland Immunochemicals, cat.MB070) and probed at 4°C overnight with antibodies against VEGF-A (1:1000; Sigma SAB2502119); cleaved mature IL-1β (1:500; Cell Signaling Technology 52718); β-Actin (1:1000, sc-47778). After multiple washes, membranes were incubated with fluorescent secondary antibodies (Thermo Fisher A21088, 1:5000; Thermo Fisher A10043, 1:5000; and LI-COR 926–32212, 1:5000). Protein bands were quantified by densitometry, using the LI-COR Imager (Odyssey CLx) and normalization to β-Actin.
9
Isolation and Characterization of Helicobacter bizzozeronii LPS
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LPS was extracted from biomass obtained after 48 h of incubation in BHI broth. Crude LPS was extracted by using the hot phenol-water method, and subsequent purification by enzymatic treatments (RNase A, DNase II and proteinase K) as described previously [6 (link)]. LPS were treated with Lysozyme to remove traces of peptidoglycan contamination [6 (link)]. After the enzymatic treatments, the LPS was precipitated at −20 °C overnight in 10 volumes of pure ethanol in presence of 0.03 M of sodium acetate and re-suspended in water and the concentration was then determined by purpald assay [20 (link)]. The LPS obtained was essentially free of proteins and nucleic acids, and it had an electrophoretic profile similar to that previously reported for the low-molecular-mass H. bizzozeronii LPS [11 (link)]. LPS was treated overnight with 6.7 U.mL−1 of neuraminidase from Clostridium perfringens (Sigma-Aldrich) at pH 6. LPS neuraminidase treated and untreated samples were loaded on 15% TRIS-Glycine SDS-PAGE (Biorad, Hercules, CA, US), run for 2 h and 50 min at constant 20 mA and then silver stained as previous described [11 (link)].
10
Stimulation and Detection of MLKL Expression
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U937 cells were seeded into 48-well plates at 60,000 cells/well and induced for 3 hours with doxycycline (20 ng/mL, 100 ng/mL or 500 ng/mL) to stimulate MLKL expression. HT29 cells were seeded into 48-well plates at 45,000 cells/well and following 12–14 hours of cell adherence, cells were induced overnight with doxycycline (20 ng/mL, 100 ng/mL or 500 ng/mL) for stimulation of MLKL expression. BMDMs were plated at 400,000 cells/well in a 24-well plate and MDFs (primary and immortalised) were plated at 30,000 cells/well in a 48-well plate. Cells were stimulated as indicated for 6 hours, except for BMDMs stimulated with LPS for 2 hours before addition of Smac mimetic Compound A for a further 4 hours. Human primary PBMCs were plated at 45,000 cells/well and stimulated for 4 hours. All cells were harvested in SDS Laemmli’s lysis buffer, boiled at 100 °C for 10–15 min, and then resolved by 4–15% Tris-Glycine SDS-PAGE (Bio-Rad). Proteins were transferred to nitrocellulose or PVDF membrane and probed with antibodies as indicated.
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