Cells were collected, washed once with cold PBS, and lysed at 4 °C for 15 min in lysis buffer [1% NP40, 10 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, protease inhibitor cocktails (Nacalai Tesque, Kyoto, Japan), 1 mM Na3VO4, and 50 mM NaF], they were then centrifuged at 15,000 rpm for 15 min at 4 °C. The supernatant was collected, and the amount of protein in the cell lysate was measured using a TaKaRa BCA Protein Assay Kit (TaKaRa Bio, Shiga, Japan) according to the manufacturer’s recommendations. The samples were separated on a 10% acrylamide gel before proteins were blotted onto a PVDF membrane (Merck, Darmstadt, Germany). After blotting, the membrane was blocked with blocking buffer (Tris-buffered saline with 0.05% Tween 20 and 5% skim milk) for 1 h at room temperature. The membrane was incubated with a primary antibody, mouse monoclonal anti-FLAG antibody (M2; Sigma-Aldrich; dilution 1:1000), an anti-fPD1 antibody in TBS-T with 0.5% skim milk at 4 °C overnight. The membrane was washed 3 times with TBS-T for 10 min at a time and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG secondary antibody (Biolegend Japan, Tokyo, Japan; dilution 1:5000) for 1 h. The membrane was imaged with an Amersham ImageQuant 800 after being washed a further three times with TBS-T for 10 min (Cytiva).
Amersham imagequant 800
Manufactured by Cytiva
Sourced in United States, Japan, United Kingdom
The Amersham ImageQuant 800 is a digital imaging system designed for the detection and analysis of fluorescent and chemiluminescent signals in biological samples. It provides high-resolution imaging and quantitative data analysis for a range of applications, including Western blotting, gel documentation, and plate-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (5 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (5 mentions)
Chemi lumi one super, by Nacalai Tesque (3 mentions)
Protease inhibitor cocktail, by Nacalai Tesque (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Protein assay kit, by Bio-Rad (2 mentions)
Nh2 pprkprrwrn s po3h2 is cooh, by Eurogentec (2 mentions)
Trans blot, by Bio-Rad (2 mentions)
Anti p42 44 phospho erk antibody, by Cell Signaling Technology (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Takara bca protein assay kit, by Takara Bio (1 mentions)
Mouse monoclonal anti flag m2 antibody, by Merck Group (1 mentions)
Ab181020, by Abcam (1 mentions)
Mouse anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
Ripa kit, by Merck Group (1 mentions)
Mab374, by Merck Group (1 mentions)
Bis tris gradient gel, by Bio-Rad (1 mentions)
Complete mini inhibitor mixture, by Roche (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
58 protocols using amersham imagequant 800
1
Western Blot Analysis of Proteins
2
Western Blot Analysis of CXCR4 Expression
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Protein was isolated from endothelial cells with a RIPA Kit (Sigma Aldrich, R0278, Burlington, MA, United States) supplemented with protease and phosphatase inhibitors. Protein concentration was determined via BCA protein assay (Thermo Fisher Scientific, 23227, Oakwood, OH, United States) per manufacturer’s instructions. Protein lysates (40 μg/lane) were loaded for probing CXCR4 (1:500 dilution; Abcam, Ab181020, Waltham, MA, United States). Following primary antibody incubation (overnight at 4°C), blots were incubated (1 h at room temperature) with a mouse anti-rabbit IgG-HRP (1:3,000 dilution; Santa Cruz, SC-2357, Dallas, TX, United States). Immunoreactive bands were detected using a western blot imaging system (Cytiva, Amersham ImageQuant 800, Marlborough, MA, United States). GAPDH was used as a loading control (1:400 dilution; Millipore Sigma, MAB374, Burlington, MA, United States).
3
Protein Extraction and Western Blot Analysis
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Single cell organoid suspensions or monolayer cells were lysed in 125–250 μL ice-cold RIPA buffer (Pierce, #89900) supplemented with 1x Complete Mini inhibitor mixture (Roche, #11836153001) and mixed on a rotator at 4°C for 30 minutes. Protein concentration was quantified using the Bio-Rad DC Protein Assay (Catalog #500–0114). 10–20 μg of total protein was separated on 4–12% Bis-Tris gradient gels (Bio-Rad) by SDS-PAGE and then transferred to nitrocellulose membranes. Antibodies used for western blots are listed in Supplementary Table 10 . Blots were developed in Amersham ECL western detection region (Cytiva, RPN2236) and imaged on a Cytiva Amersham ImageQuant 800.
4
Quantifying Radiation-Induced Protein Changes
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The FM3A cells were pretreated with different concentrations (0, 12.5, and 25 μM) of AM-18002 for 2 h and then exposed to IR at 0, 4, and 8 Gy. The next day, the cells were washed with cold PBS and lysed using lysis buffer (#TLP-121CETi, TransLab). The protein concentration was quantified using the Bio-Rad Protein Assay Kit (#5000001, Bio-Rad). Protein samples (10 μg) were loaded onto 4%–12% Bis-Tris Plus gradient gels (#NW04120, Invitrogen) and then transferred to polyvinylidene fluoride membranes (#10600100, GE Healthcare). The membranes were blocked with 5% skim milk (#232100, BD) at room temperature for 1 h and incubated with primary antibodies at 4°C overnight. Following incubation, the membranes were washed three times with Tris-buffered saline with Tween20 (TBS-T) and reprobed with secondary antibodies at room temperature for 2 h. Finally, the membranes were developed using ECL solution (#RPN2109, GE Healthcare) and visualized and captured using an Amersham ImageQuant 800 (Amersham) imaging system.
5
Nucleosome Assembly and Micrococcal Nuclease Digestion
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This refers to Supplementary Figure S5B . The nucleosome assembly reaction was carried out at 200 nM of 207 bp DNA, 200 nM xenopus octamer maleimide AlexaFluor-647 (AF647) labeled on H2B T112C (containing H3 C110A mutant) and 500 nM CAF-1, Polϵ or RPA. After the assembly reaction, the samples were diluted to a DNA concentration of 50 nM in 100 μl digestion reactions. 25U of MNase enzyme was added in a final buffer containing 50 mM Tris pH 7.9, 5 mM CaCl2. After incubation at 37°C for 10 min, the reactions were quenched with 10 μl of 500 mM EDTA, pH 8. The DNA was then purified using a modified protocol of the MinElute kit from QIAGEN. 550 μl of PB buffer and 10 μl of 3 M sodium acetate were added to each sample and they were incubated at room temperature for 10 min. At this point, 50 ng of DNA loading control (or reference band, a 621 bp DNA fragment) was added to each tube. The samples were applied to the MinElute spin column and washed as prescribed by QIAGEN. The DNA was eluted with 10 μl of water. 2.5 μl were loaded on a 6% PAGE gel. The gel was run for 45 min at 200 V in 0.5× TBE buffer at room temperature. Gels were stained with SybrGOLD for DNA and imaged on an AMERSHAM ImageQuant 800 (Cytiva).
6
Western Blot Analysis of Hippocampal Proteins
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Thirty micrograms of hippocampal proteins from mice were separated using 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) (Beyotime Biotechnology) and then transferred to a polyvinylidene fluoride membrane (PVDF) (Millipore). The membrane was washed thrice with Tween 20 (TBST) Tris‐salt solution and blocked with 5% BSA or skim milk for 1 h. The primary antibody, diluted with 5% BSA‐TBST, was then applied and incubated overnight at 4°C. Following a rinse with TBST, the membrane was incubated with HRP‐conjugated secondary antibody (Proteintech) at room temperature for 1 h. The film was exposed to enhanced chemiluminescence (ECL) (Millipore) for 30 s before evaluation using an Amersham ImageQuant 800 (Cytiva) and subsequent quantification using ImageJ software. The study used three groups of mice for experimentation, and Table 1 lists the primary antibodies.
7
Western Blot Analysis of Protein Expression
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Cells were lysed with radioimmunoprecipitation assay buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) containing 1 mM NaF and 1 mM NaVO4 for 15 min at 4 °C. Proteins in the cell lysates were resolved by SDS-PAGE and transferred to a PVDF membrane (Millipore). The blots were blocked with 5% skim milk and incubated with primary and secondary antibodies. Immunoreactive bands were detected using the West-Q PICO Dura ECL solution (GenDepot, Barker, TX, USA), Immobilon Western Chemiluminescent HRP Substrate (Millipore), and Amersham ImageQuant 800 (Cytiva, Marlborough, MA, USA). Protein bands obtained by Western blot were quantified using ImageJ software.
8
Detailed SDS-PAGE and Western Blot Protocol
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Cell lysates were obtained as described previously (Sluysmans et al., 2021 (link)) with the following modifications: SDS-PAGE was carried out at RT, and transfer onto nitrocellulose membrane was carried out at 85 V for 120 min. Blocking was realized with Tris-buffered saline (TBS) with 0.1% Tween-20 and 5% low-fat milk, and incubation with primary and secondary antibodies in the same buffer containing only 3% low-fat milk.
Chemiluminescence (ECL) was detected using Odyssey Imager (LI-COR) or Amersham ImageQuant 800 (Cytiva). Numbers on the left of immunoblots correspond to sizes in kilodaltons (kDa) of prestained markers. Uncropped blots are shown inFig. S8 .
Chemiluminescence (ECL) was detected using Odyssey Imager (LI-COR) or Amersham ImageQuant 800 (Cytiva). Numbers on the left of immunoblots correspond to sizes in kilodaltons (kDa) of prestained markers. Uncropped blots are shown in
9
SDS-PAGE and Native-PAGE Analyses
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For SDS-PAGE analysis, 4–20% SDS-PAGE Mini-PROTEAN® TGX Precast gels (4561094, Bio-Rad) were used with running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3; Bio-Rad). Samples were added to SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 25% (v/v) glycerol, 0.01% bromophenol blue, and 100 mM DTT) and denatured at 95 °C for 5 min. For Native-PAGE analysis, purified ligands were added to Native-PAGE sample buffer (75 mM Tris, 576 mM glycine, 30% (v/v) glycerol, 0.01% bromophenol blue) and run in a 4–20% SDS-PAGE Mini-PROTEAN® TGX Precast gels (4561094, Bio-Rad) with running buffer (25 mM Tris, 192 mM glycine). To assess A’-Γ:Γ’-A’ complex formation, equimolar concentrations of purified A’-Γ and Γ’-A’ ligands were first incubated at 30 °C for 1 hour and the formed complex was run in a Native-PAGE gel (Supplementary Fig. S24a ). Protein visualization was done with Coomassie Brilliant Blue G-250 (1610406, Bio-Rad). Gels were analyzed with the ImageQuant 350 (GE Healthcare) or Amersham ImageQuant 800 (Cytiva). All procedures were carried out according to the manufacturer’s protocols.
10
SDS-PAGE and Western Blot Analysis
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Cells were lysed in SDS lysis buffer (50 mM Tris-HCl pH7.5, 100 mM NaCl, 1 mM EDTA, 1% SDS, 10 mM NaF, 10 mM β-glycerophosphate, 2 mM Na3VO4). Fractions from the vesicle isolation, or cell lysates were subjected to SDS-PAGE, followed by transfer to Immobilon-P PVDF membranes (Millipore). Membranes were blocked in 1% BSA/TBS containing 0.1% Tween20 for 30 min, and treated with primary antibodies in blocking buffer overnight at 4°C, followed by treatment with HRP-conjugated secondary antibodies (Dako) for 1 h. Bands were visualized using Chemi-Lumi One Super or Chemi-Lumi One Ultra (Nacalai Tesque, 02230–30 and 11644–40) according to the manufacture’s protocol, and images were obtained using an Luminograph I quantitative Cooled CCD camera detection system (ATTO) or an Amersham™ ImageQuant™ 800 (Cytiva) as unsaturated 16-bit TIFF images. Silver staining of gels were performed using Pierce™ Silver Stain for Mass Spectrometry kit (Thermo Fisher, 24600).
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