Prrx1+ cells with or without tension force load were collected for extracting protein. Western blotting was performed with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then the proteins were transferred into a nitrocellulose membrane, and membranes were blocked by nonfat milk. After blocking, the nitrocellulose membranes were then incubated using anti- Pallidin (Proteintech, 1:500, 10891–2-AP), anti-Smad2/3 Ab (Abcam, 1:500, ab202445), anti-pSmad2/3 Ab (Abcam, 1:500, ab63399), anti-GAPDH (Proteintech, 1:1000, 110494–1-AP). The figures for western blotting were visualized using enhanced chemiluminescence reagent (Thermo Fisher Scientific, Waltham, USA) and imaged by the ChemiDoc XRS Plus luminescent image analyzer (Bio-Rad).
Chemidoc xrs plus
Manufactured by Bio-Rad
Sourced in United States, Japan
The ChemiDoc XRS Plus is a compact, high-performance imaging system designed for a wide range of life science applications. It features a high-resolution CCD camera, a UV transilluminator, and multiple filter options for capturing images of various sample types, including gels, blots, and chemiluminescent samples.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (8 mentions)
Clarity western ecl substrate, by Bio-Rad (7 mentions)
Blocking one, by Nacalai Tesque (5 mentions)
Immobilontm western, by Merck Group (5 mentions)
Chemi lumi one l, by Nacalai Tesque (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Enhanced chemiluminescence reagent, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (2 mentions)
Quantity one software, by Bio-Rad (2 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (2 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Cytiva (2 mentions)
Polyvinylidene difluoride membrane, by Pall Corporation (2 mentions)
Gradient sds polyacrylamide gel, by Bio-Rad (2 mentions)
Anti rabbit igg, by Santa Cruz Biotechnology (2 mentions)
Leupeptin, by Nacalai Tesque (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Anti rabbit igg h l, by Bio-Rad (2 mentions)
Nitrocellulose membrane, by GE Healthcare (2 mentions)
Premixed laemmli sample buffer, by Bio-Rad (2 mentions)
73 protocols using chemidoc xrs plus
1
Investigating Prrx1+ Cell Mechanobiology
2
Southern Blot Analysis of Ppsnrk2 Genomic DNA
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The genomic DNA of the Ppsnrk2 plants was extracted using PhytoPure (GE Healthcare) with an additional RNase treatment. The probes were amplified using a PCR DIG Probe Synthesis Kit (Roche Applied Science) with primers described in Suppelemtary Data 8 . Two µg of genomic DNAs were digested with restriction enzymes and were separated on 0.6% agarose gels and transferred to nylon filters (Hybond-N, GE Healthcare). The membrane was hybridized with DIG-labelled probes in DIG Easy Hyb buffer (Roche Applied Science) at 41 °C overnight. After washing, the membrane was treated once with blocking buffer for 1 h at room temperature. The membrane was reacted with Anti-Dig (Roche Applied Science) for 1 h at room temperature. Detection was carried out with CDP-Star (Roche Applied Science) for 5 min at room temperature in the dark. The signal was detected by ChemiDoc XRS Plus (BIO-RAD).
3
CRISPR-Cas9 Cleavage Assay Protocol
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crRNAs were purchased from Integrated DNA Technologies (Coralville, IA, USA). crRNA/tracrRNA was annealed in annealing buffer (50 mM Tris–HCl, 150 mM NaCl, pH 7.5). The cleavage reaction was performed by mixing crRNA/tracrRNA (30 nM) and Cas9 proteins (50 nM) in cleavage buffer (20 mM HEPES, pH 6.5, 100 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA). After 10 min incubation, annealed aptamer was added to the reaction at different concentrations. Ten minutes later, the plasmid containing the target site was added to the reaction mix (10 μl in total). The reaction was performed at 37°C for 20 min and then run in 0.7% TAE agarose gel. After electrophoresis, the gels were visualized by ChemiDoc XRS Plus (Bio-Rad, Hercules, CA, USA) imaging. The percentage of cleavage was quantified using bio-rad image lab 5.0 software.
Three independent assays were done for each in vitro cleavage experiment and error bars shown in all panels are standard deviation (SD). In the quantification of the gel image, the % of the uncleaved plasmid (unit of vertical axis in Figure1B and C ) was calculated using the following formula: percentage of uncleaved plasmid = 100 × ([uncleaved plasmid (supercoiled band)]/[uncleaved plasmid + cleaved plasmid (linear and nicked bands)].
Three independent assays were done for each in vitro cleavage experiment and error bars shown in all panels are standard deviation (SD). In the quantification of the gel image, the % of the uncleaved plasmid (unit of vertical axis in Figure
4
Western Blot Technique for Protein Analysis
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Western blotting was conducted following the method of Ozaki et al.[32] (link). Briefly, each sample was separated using SDS-PAGE and transferred onto Immobilon-P polyvinylidene fluoride (PVDF) transfer membranes (catalog number: IPVH00010; EMD Millipore, Billerica, MA, USA). The membranes were blocked with blocking buffer (10 mM sodium phosphate buffer, pH 7.4, 0.14 M NaCl, and 0.05% Tween 20 [TW-PBS] containing 1% skim milk or TW-PBS containing 5% skim milk) for 4 h at 25°C and then incubated overnight with the primary antibodies at 4°C. After washing with TW-PBS, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies overnight at 4°C. Each antibody had been diluted with the blocking buffer as follows: anti-mouse ES1 (1:1000), anti-porin (1:1000), anti-Ak2 (1:2000 or 1:1000), anti-COX4 (1:2000), anti-AIF (1:500), anti-ATP5A (1:200), and anti-beta-actin (1:10,000). After incubation and washing, the immunoreactive signals were detected using the ECL Prime Western Blotting Detection Kit (catalog number: RPN2236; Cytiva, Tokyo, Japan). The images were captured using a luminescent image analyzer (ChemiDoc XRS Plus; catalog number: 1708265J1PC; Bio-Rad Laboratories, Hercules, CA, USA).
5
Chondroitin Sulfate A Protein Detection
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Nitrocellulose membrane was prewetted with PBS and air dried 30 minutes at room temperature; 2 μL of isolated protein were directly blotted to nitrocellulose membrane. Membranes were then air dried for 30 minutes at room temperature, and blocked with 5% BSA in TBS‐T. Subsequently, membranes were incubated with anti‐chondroitin sulfate A (2H6) (Cosmo Bio, #NU‐07‐001) at 1:5000 dilution, followed by incubation with HRP‐labeled secondary antibody (TCI Chemicals, #G0417) at 1:2000 dilution. Signals were elicited by Clarity Western ECL Substrate (Bio‐Rad, #1705061), and detected using ChemiDoc XRS Plus (Bio‐Rad).
6
Western Blot Analysis of Ponatinib Treated Cells
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After serum starvation for 24 h using DMEM supplemented with 0% FBS for DHS-1 and 1% FBS for DHS-2, respectively, cells were treated with 1 µM of ponatinib for 3 h. Same amount of whole cell lysate samples extracted from these treated cells was separated by SDS-PAGE and blotted on PVDF membranes. After blocked with 5% bovine serum albumin (MilliporeSigma, USA), membranes were incubated with primary antibodies as shown in Additional file 1 : Table S5 . After incubation with primary antibodies, the membranes were washed and incubated with HRP-conjugated secondary antibody. Then, the membranes were incubated with Luminata Forte Western HRP Substrate (MilliporeSigma). Membranes were visualized using ChemiDoc XRS Plus (Bio-rad Laboratories, USA), and density of bands was analyzed by ImageJ software. Density analysis was conducted using the data obtained in triplicate.
7
Western Blot Analysis Protocol
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Cell lysates were prepared in SDS-sample buffer were separated by SDS-PAGE and electrotransferred onto polyvinylidene difluoride membranes (PVDF, Millipore). Immunodetection was performed as reported previously20 (link),34 (link),36 (link),43 (link). Sequential reprobing of membranes with a variety of antibodies was performed after the complete removal of primary and secondary antibodies from membranes in stripping buffer or inactivation of horseradish peroxidase by 0.1% NaN3, according to the manufacturer’s instructions. Results were analyzed using an image analyzer ChemiDoc XRSplus (Bio-Rad). Intensity of chemiluminescence was measured using the Quantity One software (Bio-Rad).
8
Western Blot Analysis of Cellular Proteins
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The procedure of western blot was the same as the previous paper [23 (link)]. In brief, the total protein from the cells was lysed with radio immunoprecipitation assay buffer (Solarbio Biotech). Protein concentrations were measured using BCA protein assay kit (Beyotime) according to the manufacturer’s instructions. Then, equivalent amount of protein were separated by SDS-PAGE and transferred into polyvinylidene fluoride membranes (Bio-Rad). After blocking with 5% non-fat milk for 1 h at room temperature, the membranes were incubated with primary antibodies at 4 °C overnight and then incubated with secondary antibodies for 2 h at room temperature. Bands were visualized with an ECL detection kit (Thermofisher) using ChemiDoc XRS Plus (BioRad) and analyzed using the Image J software. All primary antibodies and secondary antibodies in the study are listed as follows: anti-ACSM5 antibody (1:500, Proteintech), anti-collagen I (1:1000, Abcam), anti-collagen III (1:1000, Abcam), anti-MMP2 (1:1000, Proteintech), anti-MMP13 (1:1000, Proteintech), and anti-β-actin antibody (1:5000, Abcam).
9
Western Blot Analysis of Protein Phosphorylation
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Cell lysates that were dissolved with an SDS-sample buffer were separated by SDS-PAGE and electrotransferred onto polyvinylidenedifluoride (PVDF) membranes (Pall Corporation, Port Washington, NY, USA). To examine the phosphorylation of proteins, cells were dissolved with an SDS-sample buffer containing the phosphatase inhibitors 20 mM β-glycerophosphate, 50 mM NaF, and 10 mM sodium orthovanadate. Blots were incubated with Blocking One (03953-95, Nakalai Tesque, Kyoto, Japan) on the rocking shaker for 30 min at room temperature, and sequentially probed with primary and secondary antibodies that were diluted with 0.1% Tween 20-conaining Tris-buffered saline for 1 h at room temperature or overnight at 4 °C. Chemiluminescence was detected with the image analyzer ChemiDoc XRSplus (Bio-Rad, Hercules, CA, USA) using Chemi Lumi-One L (#07880, Nacalai Tesque) or Clarity (170-5061, Bio-Rad) as the chemiluminescence substrate. The amount of protein was quantified by measuring the signal intensities of the bands using ImageJ software (NIH, USA).
10
Cyanobacterial Protein Extraction and Western Blotting
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The protein extracts from cyanobacteria cells were prepared according to the method described by Nimura et al. (2001) (link) and were separated by SDS-PAGE on 12% polyacrylamide gels at 150 V for 90–100 min in electrophoresis buffer (25 mM Tris, 192 mM glycine, and 0.1% SDS). The gels were blotted onto 0.2 μm PVDF membranes (Bio-Rad, Hercules, CA, United States) at 2.5 A for 10 min using a Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked for 30 min with 5% skimmed milk in TBS-T [50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 0.05% Tween 20], washed, and soaked in TBS-t for 2 h at room temperature with anti-GFP (MBL, Tokyo, Japan), anti-RpoD1 rabbit antiserum (Seki et al., 2007 (link)), or anti-RbcL (Agrisera, Vännäs, Sweden) as primary antibodies. After washing, the membranes were soaked in TBS-t for 30 min at room temperature with HRP-conjugated anti-mouse (GE Healthcare, Chicago, IL, United States) or anti-rabbit antibody (GE Healthcare) as secondary antibodies. Chemiluminescence was detected with the LumiGLO Chemiluminescent Substrate KPL using ChemiDoc XRS Plus (Bio-Rad).
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