About 20 µL in vitro glutamylation reactions were performed with 2 µM SidJ/CaM, 2.5µCi α -[32 P]ATP (Perkin Elmer) in a buffer consisting of 30 mM HEPES pH 7.5, 150 mM NaCl, 10 mM MgCl2, 1 mM β-mercaptoethanol, and incubated at 37 °C for 30 min. Reactions were stopped through the addition of 5 µL of 4x SDS-PAGE sample buffer. The samples were then separated using SDS-PAGE, stained using Coomassie stain, and dried using a Model 583 gel dryer (Bio-Rad). A storage phosphor screen (GE Healthcare) was placed on the gel and exposed for 18 h, and the autoradiography signal was collected using a Typhoon FLA 7000 (GE). Raw autoradiography images of all autoAMPylation assays are available in the supplementary information file.
Model 583 gel dryer
Manufactured by Bio-Rad
Sourced in United States
The Model 583 Gel Dryer is a laboratory equipment used for drying polyacrylamide and agarose gels. It features a vacuum-assisted drying system and a temperature-controlled drying chamber to efficiently remove moisture from the gels.
Automatically generated - may contain errors
Lab products found in correlation
Storage phosphor screen, by GE Healthcare (4 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (4 mentions)
γ 32p atp, by PerkinElmer (3 mentions)
Typhoon fla 7000, by GE Healthcare (2 mentions)
Typhoon 9410 imager, by GE Healthcare (2 mentions)
Usingchef driii system, by Bio-Rad (2 mentions)
Genomic dna purification kit, by Promega (2 mentions)
Glass microfiber filter, by GE Healthcare (2 mentions)
Neg072007mc, by PerkinElmer (2 mentions)
Ls6500 liquid scintillation counter, by Beckman Coulter (2 mentions)
1225 sampling manifold, by Merck Group (2 mentions)
Complete counting cocktail 3a70b, by Research Products International (2 mentions)
Bio dot sf, by Bio-Rad (2 mentions)
Sybr gold nucleic acid gel stain, by Thermo Fisher Scientific (2 mentions)
Image lab version 6, by Bio-Rad (2 mentions)
α 32p atp, by PerkinElmer (1 mentions)
L 14 c glutamate, by PerkinElmer (1 mentions)
Express 35s protein labeling mix, by PerkinElmer (1 mentions)
Anti flag m2 monoclonal antibody, by Merck Group (1 mentions)
Protein a g agarose, by Santa Cruz Biotechnology (1 mentions)
23 protocols using model 583 gel dryer
1
In vitro SidJ/CaM Glutamylation Assay
2
Telomere Length Measurement by PFGE
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Telomere length assay was performed as described previously (Deng et al., 2007 (link)) with some
modifications. Briefly, genomic DNA was isolated from cells using a genomic
DNA purification kit (Promega) and digested with AluI and MboI. Digested
genomic DNA was purified by phenol/chloroform extraction and quantified
using a NanoDrop spectrophotometer (Thermal Scientific). Equal amounts of
digested DNA (5 μg) were separated by PFGE performed with a 1%
agarose gel at 6V/cm, switch times from 1 to 6 s, 14°C for 13 h using
CHEF-DRIII system (Bio-Rad). The resolved DNA was vacuum dried at
50°C for 90 minutes using a Model 583 Gel Dryer (Bio-Rad), in-gel
denatured and neutralized and analyzed by Southern blot. The blots were
hybridized with a 32P-labeled CCCTAA repeat probe at 42°C
overnight, then washed twice for 5 min each with 0.2 M wash buffer (0.2 M
Na2HPO4 pH 7.2, 1 mM EDTA, and 2% SDS) at room
temperature and once for 10 min with 0.1 M wash buffer at 42°C.
Relative telomere-repeat signals were determined by Typhoon 9410 Imager and
ImageQuant TL software (GE Healthcare). Signal intensity represents the
percentage decrease of average telomere signals relative to dox (−)
control.
modifications. Briefly, genomic DNA was isolated from cells using a genomic
DNA purification kit (Promega) and digested with AluI and MboI. Digested
genomic DNA was purified by phenol/chloroform extraction and quantified
using a NanoDrop spectrophotometer (Thermal Scientific). Equal amounts of
digested DNA (5 μg) were separated by PFGE performed with a 1%
agarose gel at 6V/cm, switch times from 1 to 6 s, 14°C for 13 h using
CHEF-DRIII system (Bio-Rad). The resolved DNA was vacuum dried at
50°C for 90 minutes using a Model 583 Gel Dryer (Bio-Rad), in-gel
denatured and neutralized and analyzed by Southern blot. The blots were
hybridized with a 32P-labeled CCCTAA repeat probe at 42°C
overnight, then washed twice for 5 min each with 0.2 M wash buffer (0.2 M
Na2HPO4 pH 7.2, 1 mM EDTA, and 2% SDS) at room
temperature and once for 10 min with 0.1 M wash buffer at 42°C.
Relative telomere-repeat signals were determined by Typhoon 9410 Imager and
ImageQuant TL software (GE Healthcare). Signal intensity represents the
percentage decrease of average telomere signals relative to dox (−)
control.
3
In Vitro Glutamylation Assay
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About 20 uL in vitro glutamylation reactions were performed with 2uM SidJ/CaM and 2uM SdeA, 5 mM ATP, and 50 uM (0.5 nCi) l -[14 C]glutamate (Perkin Elmer) in a buffer consisting of 30 mM HEPES pH 7.5, 150 mM NaCl, 10 mM MgCl2, 1 mM β-mercaptoethanol, and incubated at 37 °C for 30 min. Reactions were stopped through the addition of 5 uL of 4x SDS-PAGE sample buffer. The samples were then separated using SDS-PAGE, stained using Coomassie stain, and dried using a Model 583 gel dryer (Bio-Rad). A storage phosphor screen (GE Healthcare) was placed on the gel and exposed for 72 h, and the autoradiography signal was collected using a Typhoon FLA 7000 (GE). Raw autoradiography images of all glutamylation assays are available in the supplementary information file.
4
Metabolic Labeling and Radioactive Quantification
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For metabolic labeling, cells were incubated in methionine/cysteine (Met/Cys)-free DMEM (Corning, 17-204-CL) supplemented with 40 mM HEPES, 2 mM L-glutamine and a 35S-L-methionine and 35S-L-cysteine mix (PerkinElmer, NEG072007MC) for 30 min prior to cell lysis. For each ml of labeling media prepared, 0.035 mCi of the 35S Met/Cys mix was added. Where inhibitors were used, inhibitors were present during labeling. After in-well lysis in Laemmli buffer, samples were resolved by SDS-PAGE and gels were then fixed in 10% acetic acid/25% methanol solution for 30 min. The fixed gels were then dried at 80°C for 2 h using a Model 583 Gel Dryer (Biorad) and exposed to audioradiography film.
To quantify the radioactivity of 35S present in the label samples, 20 μl of radiolabeled sample was incubated with 10 μl of 10 mg/ml BSA and 1 mL of ice-cold 10% TCA solution for 30 min on ice. Precipitated proteins were vacuum filtered using a 1225 Sampling Manifold (Millipore Sigma) onto glass microfiber filters (GE Life Sciences, 1822-025), and washed twice each with ice-cold 10% TCA solution and 95% ethanol. Filter counting was performed by immersing the filters into 3 mL of Complete Counting Cocktail 3a70B (Research Products International Corp., 111154). The number of counts registered per minute (CPM) was measured using a Beckman LS 6500 liquid scintillation counter with a counting time of 5 min.
To quantify the radioactivity of 35S present in the label samples, 20 μl of radiolabeled sample was incubated with 10 μl of 10 mg/ml BSA and 1 mL of ice-cold 10% TCA solution for 30 min on ice. Precipitated proteins were vacuum filtered using a 1225 Sampling Manifold (Millipore Sigma) onto glass microfiber filters (GE Life Sciences, 1822-025), and washed twice each with ice-cold 10% TCA solution and 95% ethanol. Filter counting was performed by immersing the filters into 3 mL of Complete Counting Cocktail 3a70B (Research Products International Corp., 111154). The number of counts registered per minute (CPM) was measured using a Beckman LS 6500 liquid scintillation counter with a counting time of 5 min.
5
Circadian Clock Protein Turnover
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The half-lives of transfected FLAG epitope-tagged circadian clock proteins (BMAL1, PER2 and CRY1) in the absence (as a control) and presence of siRNAs targeting COPS7B were measured by metabolic pulse chase protein labeling according to the protocol of Zhou et al. Briefly, 72 hr post-transfection, 100 mm plates of HEK293T cells were starved of amino acids for 1 hr and metabolically labeled for 30 min with 100 μCi of Express-35S protein-labeling mix (PerkinElmer Life Sciences, Schwerzenbach, CH) in 1 ml methionine- and cysteine-free DMEM medium supplemented with 10% dialysed FCS (‘the pulse’). Sunsequently, cells were washed to remove unbound radioactive amino acids, and incubated in complete medium (‘the chase’). At indicated time points (0, 2, 4, and 6 hr), the cells were disrupted in ice-cold RIPA lysis buffer (0.02M Tris/pH7.2, 0.15M NaCl, 1% Trion X-100, 1% Na-deoxycholate, 0.1% SDS), and 1000 μg of whole cell extract were immunoprecipitated with Monoclonal ANTI-FLAG M2 Antibody from Sigma (F3165). Proteins were subjected to 9% SDS-PAGE and the gels were dried for 2 hr at 80°C by Model 583 gel dryer (BIO-RAD, Hercules, CA). Labeled proteins were visualized by autoradiography and quantitative analysis of three independent experiments for each circadian clock protein was performed with ImageStudio software.
6
Phosphorylation of GFP-PIKE-A by AMPK
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After transfection, 500 μg protein from each sample were prepared and immunoprecipitated by adding 2 μl anti-GFP antibody and 25 μl of protein A-G agarose (Santa Cruz) at 4 °C for 3 h. Phosphorylation reactions were performed with immunoprecipitated GFP-PIKE-A from 500 μg total protein and 0.1 μg of active AMPKα1β1γ1 (SignalChem) in a final volume of 50 μl 10× AMPK kinase buffer (5 mM MOPS, pH 7.2, 2.5 mM β-glycerophosphate, 1 mM EGTA, 0.4 mM EDTA, 5 mM MgCl2, 0.05 mM DTT) and 1 μl [γ-32P]ATP (Perkin Elmer). Selected reactions were carried out in the presence or absence of active AMPKα1β1γ1. After incubation at 30 °C for 30 min, the reactions were terminated by addition of 2.5 μl of 5× sodium dodecyl sulfate (SDS) buffer, and the samples were subjected to 12.5% SDS-polyacrylamide gel electrophoresis (PAGE). The gels were dried with a Model 583 Gel Dryer (Bio-Rad) and phosphorylated proteins were visualized by autoradiography.
7
Radiolabeled DNA-Protein Binding Assay
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sAvL1-451 DNA were 5′-end-labeled with [γ−32P]dATP (PerkinElmer) using T4 polynucleotide kinase (NEB) and purified from excess of radioactive nucleotides using Oligo Clean & Concentrator kit (Zymo Research) following the manufacturer’s protocols. Binding reactions were set up in 10 µl total volume in a buffer with final concentrations 100 mM KCl, 10 mM Tris, pH7.4, 0.1 mM EDTA, 0.1 mM DTT, supplied with 500 ng LightShift™ Poly (dI-dC) (Thermo Scientific). Addition of 2.5 µl of AvMBD proteins provided 5% glycerol per reaction. Proteins were first pre-incubated with non-radioactive DNA for 15 min at RT. Then, 32P-labeled DNA was added to a final concentration of 0.05 nM, and reactions were incubated for additional 30 min at RT. After supplying with 6× EMSA gel-loading solution (Thermo Scientific), samples were loaded onto 6% DNA Retardation gels. Samples were run at 90 V in 0.5× TBE buffer (44.5 mM Tris–HCl, pH 8.3, 44.5 mM boric acid and 1 mM EDTA) at 4 °C for 90 min. Gels were dried using Model 583 Gel Dryer (BioRad), exposed with phosphorimaging plate (Fujifilm), scanned on Typhoon FLA 7000, and analyzed using Image Quant TL v8.1 software.
8
Telomerase Activity Quantification
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Telomerase activity was measured using the TRAPeze kit (Millipore) per manufacturer's directions. To prevent interference by PCR inhibitors in the cell extracts, samples were phenol-chloroform extracted after template elongation and before PCR amplification. Products were separated on 10% SDS-PAGE gels and dried on a BioRad Model 583 gel dryer. Gels were exposed to phosphor screens (GE) overnight and imaged on a Typhoon 9410 variable mode imager (GE). Negative control samples include no cell extract, while positive controls represent a manufacturer-provided telomerase-positive cell extract.
9
Metabolic Labeling and Radioactive Quantification
10
In vitro Kinase Assay for NbCK2α and HvCK2α
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Soluble cytoplasmic protein extracts of healthy N. benthamiana leaves were used for in vitro kinase assays according to the protocol described by Hung et al. (2014) (link) and Vijayapalani et al. (2012) (link). Phosphorylation reactions were performed with the N. benthamiana soluble protein extracts or with purified recombinant NbCK2α and HvCK2α subunits. Assays were performed with 1 μg of N. benthamiana soluble protein extracts, or 0.1 μg of recombinant CK2α, and 1 μg of purified XJTGB1 protein or its mutants in a final volume of 10 μl of 25mM Tris/HCl (pH 7.4), 10mM MgCl2, and 1 μl [γ-32P]ATP or GTP (10 μCi, ~3000 Ci mmol–1; Perkin Elmer). Selected reactions were carried out in the presence or absence of heparin, and various concentrations of unlabelled ATP or GTP. Negative controls contained no TGB1 protein or 500 and 1000ng of bovine serum albumin. After incubation at 30 °C for 30min, the reactions were terminated by addition of 2.5 μl of 5× SDS buffer, and the samples were subjected to 12.5% SDS-PAGE. The gels were dried with a Model 583 Gel Dryer (Bio-Rad) and phosphorylated proteins were visualized by autoradiography.
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