To detect the holo form of Yhb1, cells were resuspended in PBS containing a mixture of protease inhibitors and disrupted using glass beads in a BioSpec Mini-Beadbeater. These protein extracts were centrifuged (3000 rpm for 5 min) and 20 μg of protein were loaded in polyacrylamide gels and separated in native conditions (without SDS). After electrophoresis, gels were directly imaged in a ChemiDoc XRS to detect Flavin fluorescent signal (Excitation: Blue Epi Illumination; Emission: 530/28 filter) or transferred to a PVDF membrane, washed once with TBS buffer and incubated with HPR substrate to detect heme peroxidase activity. The chemiluminescent signal was acquired in a ChemiDoc XRS.
Chemidoc xr
Manufactured by Bio-Rad
Sourced in United States, United Kingdom, China, Italy, Germany, France, Japan, Switzerland, Netherlands, Canada
The ChemiDoc XRS is a compact and versatile imaging system designed for various life science applications. It captures high-quality digital images of gels, blots, and other samples illuminated by different light sources. The system features automated image acquisition and analysis capabilities to support a range of experimental workflows.
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Lab products found in correlation
Image lab software, by Bio-Rad (180 mentions)
Pvdf membrane, by Merck Group (146 mentions)
Quantity one software, by Bio-Rad (69 mentions)
Pvdf membrane, by Bio-Rad (66 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (56 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (54 mentions)
Protease inhibitor cocktail, by Roche (46 mentions)
Bca protein assay kit, by Beyotime (45 mentions)
Nitrocellulose membrane, by Bio-Rad (45 mentions)
Ripa lysis buffer, by Beyotime (41 mentions)
Clarity western ecl substrate, by Bio-Rad (39 mentions)
Image lab, by Bio-Rad (39 mentions)
Protease inhibitor cocktail, by Merck Group (39 mentions)
Polyvinylidene fluoride membrane, by Merck Group (36 mentions)
Image lab 3, by Bio-Rad (34 mentions)
Ripa buffer, by Merck Group (30 mentions)
Ripa buffer, by Thermo Fisher Scientific (29 mentions)
Ripa buffer, by Beyotime (27 mentions)
Trans blot turbo transfer system, by Bio-Rad (27 mentions)
Polyvinylidene difluoride membrane, by Merck Group (26 mentions)
2 288 protocols using chemidoc xr
1
Detection of Yhb1 protein isoforms
2
Peptide-Mediated Cas9 Nanocomplex Formation
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Peptide-mediated complexation was determined by performing a fluorescence-based electrophoretic mobility shift assay by using a non-stained and non-denaturing 1.5% agarose gel in TAE buffer with a pH of 8.3. For this, Cas9-GFP (Merck), ATTO-550-labeled tracrRNA, unlabeled crRNA (IDT), Alexa-647-labeled HDR template (IDT) and unlabeled LAH5 peptides were used. Labeled components and unlabeled LAH5 peptides were formulated at various molar ratios as described above. All labeled components were diluted such that the final concentration reached 1 µM when loaded on gel. All formulated nanocomplexes were adjusted to a total volume of 10 µL by adding nuclease-free water. Then, 2 µL of 40% Glycerol was added, after which samples were loaded into the wells of the gel. The agarose gel was run at 170 V for 15 min. Gel images were captured by ChemiDocTM XRS+ (Bio-Rad, Lunteren, The Netherlands), and fluorescent images were merged and analyzed with ChemiDocTM XRS+ (Bio-Rad) with Image lab software version 5.0.
3
Western Blot Analysis of Histone Modifications
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Protein samples were taken up in sample buffer (sample final concentration 40 mM Tris-HCl, pH 6.8, 1% SDS, 5% glycerol, 0.0125% bromophenol blue, 1% β-mercaptoethanol) and incubated for 5 minutes at 95°C. Proteins were run on a 4–12% SDS-PAGE in MES running buffer for H2A or H2AK119ub analysis or MOPS running buffer for analysis of all other proteins. Samples were transferred to a 0.45 μm nitrocellulose blotting membrane. The membrane was stained using ponceau staining and imaged using the Bio-Rad ChemiDoc XRS+ with Image Lab version 5.1 build 8. Membranes were subsequently developed with the appropriate antibodies and Clarity Western ECL (Bio-Rad) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) and scanned using the Bio-Rad ChemiDoc XRS+ and Image Lab version 5.1 build 8. Immunoblot bands were quantified using Image Lab version 6.0.1 build 34 and visualized using R version 4.0.2 with Rstudio version 1.3.1093. For the immunoprecipitations showing the BAP1-HAT1 interaction, nitrocellulose membranes were developed with appropriate antibodies and scanned on Odyssey Imaging Sytem (LI-COR Biosciences).
4
Western Blot Analysis of His-tagged Proteins
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Western blot performed as described in [55] (link). Image acquisition using a ChemiDoc™ XRS + (BioRad), and densitometry analysis preformed using ImageJ [59] , [60] (link). Western blots were performed in Transfer buffer (25 mM Tris, 200 mM glycine, 20% methanol) under standard conditions (90 V, 1 h). The protein-blotted polyvinylidene difluoride (PVDF) membrane was blocked overnight in 10% (w/v) milk in 1 × TBST (10 mM Tris–HCl pH 7.5, 100 mM NaCl, 1% (v/v) Tween 20). The membrane was probed with Anti-His6 antibody (INDIA) and developed by incubating with SuperSignal™ West Pico Chemiluminescent Substrate (Thermo Scientific) and exposing to film. Image acquisition using a ChemiDoc™ XRS + (BioRad), and densitometry analysis performed using ImageJ [59] , [60] (link).
5
Western Blot Protein Analysis
6
Quantitative Western Blot Analysis of Cell Signaling
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Cells were lysed in RIPA Lysis Buffer (Beyotime, P0013B) containing protease inhibitors and phosphatase inhibitors. Protein concentration was measured by a BCA kit (Beyotime, P0012S). 1.5 × 106 cells per group were lysed and about 20 μg proteins were loaded for SDS-PAGE. After proteins were transferred to a PVDF membrane, skim milk (5%) was used for blocking and then stained with the following antibodies: Plakoglobin, ZO-1, PD-L1, β-actin (Proteintech, 66009–1-Ig, mouse monoclonal), CEACAM6 (ABclonal), Integrin β1(Cell Signaling Technology, 4706P, rabbit polyclonal), Integrin β5 (Cell Signaling Technology, 4708P, rabbit polyclonal), vimentin (Santa Cruz Biotechnology), Anti-HIF-1α (Abcam, ab15106, rabbit polyclonal), GAPDH (Bioworld, AP0063, rabbit polyclonal). Goat anti-Rabbit IgG(H&L)-HRP (Bioworld, BS13278) and Goat anti-Mouse IgG(H&L)-HRP (Bioworld, BS12478) were used as the second antibodies. The blots were scanned by Gel Imaging System (Bio-Rad, ChemiDoc XRS + , USA) with ECL western blotting kit. Protein expressions were quantified using Image lab software (Bio-Rad, ChemiDoc™ XRS + , USA).
7
Western Blot Analysis of mTOR and AKT
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For Western blot analyses, protein samples were prepared and analyzed as described before [4 (link)]. Briefly, proteins were isolated using RIPA buffer, and 25 µg per sample was separated by SDS-PAGE using 4–15% Criterion TGX gradient gels (BioRad Laboratories, Munich, Germany). Proteins were transferred to a nitrocellulose membrane by wet blotting and protein imaging was performed using UV transillumination on a Chemidoc XRS (Bio-Rad Laboratories). For the detection of target protein-specific primary antibodies [mTOR (7C10) Rabbit mAb, # 2983, 1:1500; Phospho-mTOR (Ser2448) (D9C2) XP Rabbit mAb, # 5536, 1:2000; AKT (pan) (C67E7) Rabbit mAb, # 4691, 1:10,000; Phospho-AKT (Ser473) (D9E) XP Rabbit mAb, # 4060, 1:5000, each Cell Signaling Technology, Danvers, MA, USA] and a horseradish peroxidase-conjugated secondary antibody (anti-rabbit IgG, HRP-linked Antibody, Cell Signaling, #7074, 1:5000) were used. The relative protein abundance was visualized by enhanced chemiluminescence detection using an image-capture system (Chemidoc XRS, BioRad Laboratories). For normalization, the total protein loading was detected by the UV transillumination of membranes.
8
Flavivirus Antigen Detection in A549 Cells
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Overnight, A549 cells in 12-well plates were infected with the Neudoerlf TBEV strain at an MOI of 0.1. After 2 h, the medium was collected and fresh medium with different concentrations of compounds or DMSO was added for 48 h. Cells were lysed at 4 °C for 1 h with TNET buffer (20 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100). Proteins were separated by SDS–PAGE under non-reducing conditions, transferred to PVDF membranes, and detected with a specific monoclonal anti-Flavivirus group antigen antibody (4G2) (1:1500 dilution), rabbit monospecific polyclonal serum raised against prM protein (1:1500 dilution), or anti-actin antibody (1:1000 dilution) as primary antibodies. Anti-rabbit or anti-mouse peroxidase (HRP)-conjugated secondary antibodies (diluted 1:2000) were used as secondary antibodies. Antigen–antibody complexes were detected using a Super Signal West Pico Substrate system (Pierce, Dallas, TX, USA) using Chemidoc XRSþ (BioRad, Hercules, CA, USA) and analyzed.
9
Western Blotting for FGFR2 Signaling
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Western blotting was performed as previously reported27 (link) with the following primary antibodies: FGFR2 (D4L2V), phospho-FRS2α (Tyr436) and GAPDH (14C10) (Cell Signalling Technology, MA, USA), phospho-FGFR2 (pSer782) (TermoFisher, MA, USA) and FRS2α (Abcam, CB, UK). After probing for phosphorylated proteins, membranes were stripped by Re-Blot Plus Western Blot Recycling Kit (Chemicon International, Temecula, Calif) and incubated with antibodies against the corresponding total protein. GADPH was used for equal protein loading. Digital images of X-ray films were captured by ChemiDoc XRSþ (Image Lab Software, Bio-Rad).
10
Western Blot Protein Analysis
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Precipitated cells were washed with cold PBS and lysed with RIPA buffer containing 1% protease and phosphate inhibitor cocktail (P8340; Sigma‒Aldrich, USA). After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS‒PAGE), proteins were transferred to polyvinylidene fluoride (PVDF) membranes. Then, the membranes were cut into strips and incubated with primary antibodies at 4 °C overnight and were then incubated with secondary antibodies. We examined protein expression using an Odyssey fluorescence scanner (ChemiDoc XRSþ, Bio-Rad). All antibody information is provided in Table S9 .
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