In‐gel kinase activity assays were performed as described previously (Liu et al., 2017 (link)) on apple calli grown on control medium (MS agar with 3% sucrose) or on the same medium supplemented with 6% mannitol or 100 µM ACC. Radioactivity was quantified using a Typhoon 9410 imager (Molecular Dynamics, GE Healthcare, Pittsburgh, PA, USA).
Typhoon 9410 imager
Manufactured by GE Healthcare
Sourced in United States, United Kingdom
The Typhoon 9410 is a versatile fluorescence and chemiluminescence imager designed for a range of life science applications. It provides high-resolution, high-sensitivity imaging for a variety of gel and blot-based detection techniques.
Automatically generated - may contain errors
Lab products found in correlation
Imagequant 5, by GE Healthcare (7 mentions)
Imagequant tl, by GE Healthcare (7 mentions)
Wizard genomic dna purification kit, by Promega (3 mentions)
Genescreen plus hybridization membrane, by PerkinElmer (3 mentions)
Genomic dna purification kit, by Promega (3 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (3 mentions)
α sarcin, by GE Healthcare (3 mentions)
Model 583 gel dryer, by Bio-Rad (2 mentions)
Usingchef driii system, by Bio-Rad (2 mentions)
Imagequant, by GE Healthcare (2 mentions)
Odyssey infrared imager 9120, by LI COR (2 mentions)
Click it hpg alexa fluor 488 protein synthesis assay kit, by Thermo Fisher Scientific (2 mentions)
α sarcin, by Santa Cruz Biotechnology (2 mentions)
Superscript 3, by Thermo Fisher Scientific (2 mentions)
Bradford assay, by Bio-Rad (2 mentions)
Mouse α sod3, by Novus Biologicals (2 mentions)
Rabbit α sod1, by Abcam (2 mentions)
Rabbit α sod2, by Novus Biologicals (2 mentions)
Alexafluor 488 goat α rabbit igg, by GE Healthcare (2 mentions)
Alexafluor 488 goat α mouse igg1, by GE Healthcare (2 mentions)
65 protocols using typhoon 9410 imager
1
In-gel Kinase Activity Assays
2
Oligonucleotide Annealing and Gel Electrophoresis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single-stranded oligonucleotides (Y6rep6’, Y2rep2’ and Y2) were 5′end-labelled with [γ32P]ATP using a T4 polynucleotide kinase (NEB). DNA samples were prepared in a cacodylic acid buffer (10 mM) at pH 7.2 (LiOH) containing NaCl or KCl (100 mM) and MgCl2 (10 mM) at a strand concentration of about 15 nM of radiolabelled oligonucleotides (Y6rep6’, Y2rep2’ and Y2) and 1.5 μM of non-radiolabelled oligonucleotides (HpE6rep6, R6rep6, HpE2rep2, R2rep2 and HpE2). Sample annealing was carried out as indicated in figure legends. Polyacrylamide gels (12%, acrylamide:bisacrylamide mass ratio of 19:1) were prepared in a TBE buffer, supplemented with NaCl or KCl (20 mM) and with MgCl2 (10 mM). Electrophoresis was run in a TBE buffer, supplemented with NaCl or KCl (20 mM) and with MgCl2 (10 mM), in a cold room, at 3 W/gel, for about 3 h. The temperature of the gel during migration was about 15 °C. Gels were dried and exposed to Phosphorimager screens and screens were scanned with a Typhoon 9410 Imager (Molecular Dynamics). In PAGE experiments shown in Fig. 2c and in Fig. S5 , oligonucleotides were not radiolabelled and they were detected by UV-shadow at 254 nm with a G:BOX (Syngene).
3
Telomere Length Analysis by Southern Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TRF analysis was conducted as previously described in ref. 22 (link). In brief, genomic DNA was prepared using Wizard genomic DNA purification kit (Promega) as manufacturer’s instruction. For telomere length and Southern blot analysis, genomic DNA (~ 5 μg) was digested with AluI + MboI restriction endonucleases, fractionated in a 0.7% agarose gel, denatured, and transferred onto a GeneScreen Plus hybridization membrane (PerkinElmer). The membrane was cross-linked, hybridized at 42 °C with 5′-end-labeled 32P-(TTAGGG)4 probe in Church buffer, and 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. The images were analyzed by Phosphor-imager, visualized by Typhoon 9410 Imager (GE Healthcare), and processed with ImageQuant 5.2 software (Molecular Dynamics).
4
Rapid Kinetic Analysis of Ubiquitin Transfer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Reactions were assembled in two separate mixtures: an E1/E2 mix that contained excess unlabeled peptide (tube 1) and an SCF-32P-labeled substrate mix (tube 2; see Supplementary file 2 for concentrations). Following addition of E2 and/or ARIH1 to tube 1 already containing reaction buffer (30 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM MgCl2, 2 mM DTT and 2 mM ATP) and ubiquitin, each mix was incubated for at least 8 min while being loaded into separate sample loops on a KinTek RQF-3 quench flow instrument. Reactions were initiated by bringing the two mixes together in drive buffer (30 mM Tris-HCl (pH 7.5), 100 mM NaCl), and then quenched at various time points in reducing 2x SDS-PAGE loading buffer (100 mM Tris-HCl (pH 6.8), 20% glycerol, 30 mM EDTA, 4% SDS, and 4% beta-mercaptoethanol). Each reaction was performed at least in duplicate, and time points were resolved on 18% polyacrylamide SDS-PAGE gels. Autoradiography was performed using a Typhoon 9410 Imager and Image Quant software (GE Healthcare). Each product species was quantified as a fraction of the total signal of its respective lane. The rates of ubiquitin transfer were determined by fitting to analytical closed-form solutions (Pierce et al., 2009 (link)) using Mathematica.
5
In vitro Mps1 Kinase Assay
6
Western Blot Protein Detection Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein samples were resolved in 10% SDS-PAGE gel electrophoresis before being transferred to an Immobilon FL membrane (Millipore.) Membranes were blocked in Tris buffered saline solution containing 1% Tween-20 and 0.25% bovine skin gelatin. The membranes were incubated with primary antibodies overnight at in the blocking solution. After removing the primary antibody and washing the membrane in the blocking solution, the secondary antibodies were applied in the blocking solution for 1 hour. Membranes were washed again in the blocking solution and imaged on a Typhoon 9410 imager (GE) after washing with PBS.
7
Southern Blot Analysis of Telomere Length
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For telomere length analysis we carried out Southern blots on HEK293T cells stably expressing WT and mutant 1xFlag-POT1 and an shRNA targeting the 3′UTR of the endogenous POT1. Genomic DNA from cells at various passages was purified using a QIAamp DNA mini kit (Qiagen) and 10 μg of genomic DNA was then digested with AluI+MboI restriction endonucleases. 5′ 32P-labelled GeneRuler 1 kb Plus DNA ladder (Thermo) and 2 μg of digested DNA was then fractionated in a 0.7% agarose gel, denatured and transferred onto a GeneScreen Plys hybridization membrane (Perkin Elmer) overnight. The membrane was cross-linked, hybridized at 42 °C with 5′-end-labaled 32P-(TTAGGG)4 probe in Church buffer (0.5 N Na2HPO4, pH 7.2, 7% SDS, 1% BSA, 1 mM EDTA) overnight and then washed three times for 20 min each with wash buffer (0.2 M Na2HPO4, pH 7.2, 1 mM EDA, and 2% SDS) at 37 °C. The membrane was exposed to a phosphorous storage plate overnight and visualized by Typhoon 9410 Imager (GE Healthcare). Telomere length was calculated using the software TeloTool (MATLAB)45 (link).
8
Telomere Length Assay by Southern Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For telomere length assay39 (link), genomic DNA was isolated using genomic DNA purification kit (Promega) and digested with AluI and MboI. Equal amounts of digested DNA (~4 μg) were separated by 0.7% agarose gel electrophoresis in 1× TBE, denatured, and transferred to a GeneScreen Plus membrane (PerkinElmer). The blot was crosslinked, hybridized at 42 °C with 5′-end-labeled 32P-(TTAGGG)4 probe in Church buffer, and 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. The images were analyzed by Phosphor-imager, visualized by Typhoon 9410 Imager (GE Healthcare), and processed with ImageQuant 5.2 software (Molecular Dynamics).
9
2D-DIGE Protein Profiling Protocol
10
ChIP Assay for Telomere and Subtelomeric Region Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ChIP assays were performed with the protocol provided by Millipore with minor modifications as described previously (Deng et al. 2009 (link)). Briefly, LCLs were crosslinked in 1% formaldehyde with shaking for 15 min, and DNA was sheared to between 200- and 400-bp fragments by sonication with a Diagenode Bioruptor. Quantification of ChIP DNA at subtelomeric regions was determined using quantitative PCR (qPCR) with the ABI 7900 sequence detection system (Applied Biosystems). qPCR was performed in triplicates from three independent ChIP experiments, and PCR data were normalized to input values. Primer sequences used for qPCR were designed using Primer Express (Applied Biosystems), and listed in Supplemental Table 10. Each primer sets was validated by using melting curve analysis, in which one major dissociation peak was observed. ChIP DNA at telomeres was assayed by dot blotting with γ-[32P]ATP-labeled probes specific for telomere (4 × TTAGGG) or Alu repeats (cggagtctcgctctgtcgcccaggctggagtgcagtggcgcga). After hybridization, the blot was developed with a Typhoon 9410 imager (GE Healthcare) and quantified with ImageQuant 5.2 software (Molecular Dynamics). Antibodies used in ChIP assay include rabbit polyclonal antibodies to CTCF (Millipore 07-729) and RAD21 (abcam ab992). Rabbit antibodies to TERF1 and TERF2 were generated against recombinant protein and affinity purified.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!