For detection of total protein carbonylation, wild-type cells were grown to mid-logarithmic phase (“growth”), washed twice, and cultured in 2% potassium acetate for 2.5 h (“starvation”). Growing cells were also subjected to 1 mM H2O2 treatment for 1 h as a positive control. Subsequently, cells were lysed by bead beating in carbonylation buffer (20 mM Na-phosphate, pH 6, 1 mM EDTA, and 6 M urea, plus protease inhibitors), and cell extracts were cleared by centrifugation at 10,000 g for 10 min at 4°C. Supernatants were treated with 1% streptomycin sulfate for 5 min to remove nucleic acids and cleared again by centrifugation at 10,000 g for 5 min at 4°C. Extracts were diluted to 1 µg/µl in carbonylation buffer, and 200 µl was labeled with 50 mM FTC (Fluka) for 2 h in the dark. Proteins were precipitated with TCA, and unbound FTC was removed by washing five times in ice-cold ethanol/ethyl acetate (1:1). Protein pellets were resuspended in 50 µl dissolving buffer (20 mM Na-phosphate, pH 8, 1 mM EDTA, and 8 M urea), and samples were prepared with 5× sample buffer without bromophenol blue for separation in a 12% SDS-acrylamide gel by running 5 µg of protein per lane in two separate gels. Total protein was visualized in one gel by silver staining, while fluorescent carbonylated proteins were visualized in the second gel using an Amersham Biosciences Typhoon scanner.
Typhoon scanner
Manufactured by Cytiva
Sourced in United States, United Kingdom
The Typhoon scanner is a high-performance fluorescence and phosphor imaging system designed for a wide range of life science applications. It captures images with high sensitivity, resolution, and dynamic range, making it suitable for various types of gel and blot analyses.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Cell Signaling Technology (2 mentions)
Simplyblue safestain, by Thermo Fisher Scientific (1 mentions)
Sc 134042, by Santa Cruz Biotechnology (1 mentions)
Rnase a, by Qiagen (1 mentions)
Vimentin, by Santa Cruz Biotechnology (1 mentions)
N cadherin, by Santa Cruz Biotechnology (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
P akt, by Santa Cruz Biotechnology (1 mentions)
P pi3k, by Santa Cruz Biotechnology (1 mentions)
E cadherin, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)
Typhoon imager, by GE Healthcare (1 mentions)
Cellytic m reagent, by Merck Group (1 mentions)
P eif2α, by Thermo Fisher Scientific (1 mentions)
Actin, by Merck Group (1 mentions)
Trans blot sd semi dry transfer cell, by Bio-Rad (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Ecl chemiluminescence system, by Cytiva (1 mentions)
Mouse anti β catenin, by Santa Cruz Biotechnology (1 mentions)
38 protocols using typhoon scanner
1
Quantifying Protein Carbonylation in Yeast
2
Purification and EMSA of Hox Protein Complex
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Ubx protein was His-tagged and purified from E. coli (BL21 or BL21pLysS; Agilent) 4 hours of induction with isopropyl-B-D-thiogalactopyranoside (IPTG) using Co-chromatography. Exd (in pET9a) and HthHM (in pET21b) were co-expressed and co-purified, through the His-tag attached to HthHM, in E. coli (BL21) in the same way as Ubx recombinant protein and used as a complex for all EMSAs. Protein concentrations were determined by the Bradford assay and then confirmed by SDS/PAGE and Blue Coomassie analysis (SimplyBlue SafeStain, Invitrogen). EMSAs were carried out as previously described ([47 (link)]). UbxIa was used at 250 ng/lane (high concentration) and 200 ng/lane (low concentration) and Exd/HthHM was used at 150 ng/lane. Sequences for probes used are as follows–abxWT: CCTCGTCCCACAGCTcgaatatattttataacccggagcaaatgcagcca; abxCluster1Kill: CCTCGTCCCACAGCTcgaatccccttccccacccggagcaaatgcagcca. Uppercase is linker sequence. DNA binding was observed using phosphoimaging as detected by a Typhoon Scanner (Amersham).
3
In-vivo Protein Crosslinking and Lysis
4
Stau1 Binding to HCV RNA Motifs
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We incubated 500 ng of purified Stau1 with Cy5-labeled HCV 3′-NTR or 5′-NTR in the presence or absence of unlabeled RNA competitor corresponding to 3′- or 5′-NTR. The incubation buffer contained 50 mM Tris HCl (pH7.8), 1 mM DTT, 1 mM MgCl2 and 0.01% bovine serum albumin in a final volume of 20–30 μl. After 20 min of incubation on ice, we UV irradiated the mixture (Spectrolinker XL-1000; Spectronics at 360 mJ/cm2. We treated the irradiated samples with RNase A (0.1 μg/μl; Qiagen, (Valencia, CA, USA) for 15 min at 37°C and resolved the crosslinked RNA–protein complexes on 8% sodium dodecyl sulfate (SDS) polyacrylamide gel. The labeled RNA–protein complex was visualized using a Typhoon scanner (Amersham). To confirm the Stau1-RNA crosslinked complex in the cell lysate, we transferred the complex onto a nitrocellulose membrane (Schleicher and Schuell Bioscience, (New Hampshire, USA) and western blotted for Stau1 using anti-Stau1 antibody (SC-134042, Santa Cruz Biotechnology).
5
Western Blot Analysis of Hepatocellular Carcinoma
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HCC cells were washed twice with ice-cold phosphate-buffered saline (PBS) and prepared with RIPA buffer. For Western blot analysis, 30 μg of protein per lane was separated by 12% SDS-PAGE electrophoresis and transferred to nitrocellulose membranes (Amersham, Little Chalfont, UK). The blot was blocked with 5% nonfat milk for 3 h at room temperature, incubated with appropriate polyclonal primary antibodies (SLC34A2, E-cadherin, N-cadherin, vimentin, p-PI3K, PI3K, p-AKT, AK, and GAPDH; from Santa Cruz Biotechnology, Santa Cruz, CA, USA) in TBST (100 mM Tris-HCl, pH 7.5, 0.9% NaCl, 0.1% Tween 20) overnight at 4°C. The membranes were washed and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody. The target protein was visualized by enhanced chemiluminescence reagent (Pierce, Rockford, IL, USA), and the fluorescence was scanned using a Typhoon scanner (Amersham Biosciences, Piscataway, NJ, USA).
6
In Vitro Phosphatidylserine Synthesis Assay
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The in vitro assays were performed essentially as described previously (Kojima et al., 2016 (link); Tamura et al., 2019b ). Specifically, we isolated heavy membrane fractions (12,000 × g pellet) from yeast cells cultured in SCD media. The isolated membrane fractions were incubated at 30°C for a period of time. in assay buffer (300 mM sucrose, 20 mM Tris-HCl, pH 7.5, 40 mM KCl, 2 mM CTP, 1 mM S-adenosylmethionine, 0.1 mM MnCl2, 2 mM MgCl2) in the presence of 2 μ Ci/mL[14C(U)]-L-serine. To stop PS synthesis and its transport reactions, we added 900 μ L of 2:1 chloroform/methanol to 100 μ of the samples and vortexing for 15 min. Then, 200 μ L of 0.1M KCl, 0.1 M HCl was added to the samples and further vortexed for 15 min at room temperature. After 5 min spin, the organic phase was collected and dried under N2 gas. The resulting lipids were dissolved in chloroform and subjected to the analysis by TLC followed by radioimaging with an Amersham Typhoon scanner.
7
RNA-Protein Binding Affinity Determination
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EMSAs were performed in a final reaction volume of 20 μl in EMSA buffer composed of 300 mM NaCl, 20 mM HEPES pH 8.0, 2 mM DTT, 4% (vol/vol) glycerol, and 3 mM MgCl2. The final protein concentration ranged from 0 to 16 μM, with a constant final concentration of fluorescence-labeled RNA 8 nM and 10 μg/ml of competitor yeast tRNA. The 24-mer RNA fragment (CGG)8 was labeled on its 5′-end with Cy5 fluorophore (Eurofins Genomics). After incubation of the reaction for 30 min at RT, 5 μl of the samples were loaded onto the 6% TBE (Tris-Borate-EDTA) polyacrylamide gel and run for 45 min at 100 V. The fluorescence measurements were performed using an Amersham Typhoon scanner and software v 2.0.0.6. The intensities of the bands observed on the scanned EMSAs gels were quantified with FUJI ImageJ software (v 1.53C).
8
Quantification of eIF2α Phosphorylation
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Transfected cells were grown in 6-well plates and harvested for protein extraction. Cells were lysed using the CelLytic M reagent (Sigma-Aldrich, St. Louis, MO, USA) and scraped from the plates. Lysates were separated in 12% NuPAGE Bis-Tris gels (Invitrogen) and transferred to the PVDF membrane using Trans-Blot SD semi-dry transfer cell (BioRad, Hercules, CA, USA). Membranes were blocked for 2 h using 5% dry milk in TBST (0.02 M Tris-HCl, 0.9% NaCl, 0.05% Tween 20, pH 7.6). Polyclonal antibody against phosphorylated eIF2α (p-eIF2α) (Invitrogen), actin (Sigma) and mouse anti-c-myc monoclonal antibody was diluted in 2.5% dry milk in TBST and incubated overnight at 4 °C. Horseradish peroxidase (HRP) conjugated anti-rabbit or anti-mouse antibody (Cell Signaling, Danvers, MA, USA) diluted 1:2000 were added and incubated for 1 h. Final detection was achieved using the ECL Plus™ Western Blotting (WB) detection reagents and a Typhoon scanner (Amersham Biosciences, Little Chalfont, UK).
Quantification of eIF2α phosphorylation after transfection of pcDNA-wtPKR and pcDNA-mutPKR in EPC (2 experiments) and AGK cells (1 experiment) was done at 16, 24, and 40 h post transfection. The amount of p-eIF2α measured by densitometry (Typhoon Imager, GE Healthcare, Chicago, IL, USA) was quantified with ImageJ software, and the value was normalized against β-actin levels.
Quantification of eIF2α phosphorylation after transfection of pcDNA-wtPKR and pcDNA-mutPKR in EPC (2 experiments) and AGK cells (1 experiment) was done at 16, 24, and 40 h post transfection. The amount of p-eIF2α measured by densitometry (Typhoon Imager, GE Healthcare, Chicago, IL, USA) was quantified with ImageJ software, and the value was normalized against β-actin levels.
9
Protein Expression Analysis by Western Blot
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Cells or tumor tissues were homogenized and lysed with RIPA lysis buffer [100 mM NaCl, 50 mM Tris–HCl (pH 7.5), 1% Triton X-100, 1 mM EDTA, 10 mM β-glycerophosphate, 2 mM sodium vanadate, and protease inhibitor]. Equal amounts of protein samples were separated by 10% SDS-PAGE and electrotransferred onto polyvinylidene difluoride membranes (Millipore, Boston, MA, USA). The membranes were then blocked with 5% nonfat milk in Tris-buffered saline containing 0.2% Tween 20 (TBST; Invitrogen) and then incubated with specific primary antibodies at 4°C overnight. The antibodies were as follows: mouse anti-TIPE2, mouse anti-E-cadherin, mouse anti-N-cadherin, mouse anti-β-catenin, mouse cyclin D1, mouse c-myc, and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) and visualized using the ECL chemiluminescence system (Amersham Biosciences, Piscataway, NJ, USA). The fluorescence was scanned using a Typhoon scanner (Amersham Biosciences).
10
Southern Blot Detection of Targeted Ighg3 Locus
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Murine embryonic stem cell DNA was screened for single-copy targeted insertion of T2ACre in the Ighg3 locus by digesting DNA with BglII restriction endonuclease, transferring agarose-gel run digested DNA onto a nitrocellulose membrane, and hybridizing membrane with P-32 labeled DNA probe specific for the 5’ UTR of Ighg3 (forward primer: 5’-TGAGCCAGGGTAAGTGGGAGTATG-3’; reverse primer: 5’-ATGAGGTGCAGAGTGGCTACAGG-3’) and the Neomycin resistance gene (forward primer: 5’-ACCGTAAAGCACGAGGAAGCG-3’; Reverse primer: 5’-GCACGCAGGTTCTCCGGC-3’). Southern blots were visualized with the use of a Typhoon scanner (Amersham Biosciences).
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