To construct the sre1 mutant, the gene-specific knock-out (KO) cassette was prepared by overlapping polymerase chain reaction (PCR) using primers listed in Supplementary Table S2 with the wild-type genomic DNA and the plasmid pCH233 as templates. The constructed KO cassette was introduced into the wild-type strain by biolistic transformation as previously described [18 (link)]. Replacement of the wild-type SRE1 coding region with the KO cassette containing the nourseothricin acetyltransferase (NAT) gene in the sre1 mutant was confirmed by PCR. The deletion of SRE1 in the mutant was also confirmed by Southern blot analysis using the genomic DNA samples digested with HindIII and SpeI restriction enzymes (Supplementary Figure S1 ). The digested DNA fragments were separated in an agarose gel and were transferred to an UltraBind transfer membrane (Pall-Gelman Laboratory, Washington, NY). The gene-specific probe was amplified by PCR from the wild-type genomic DNA using the primers Sre1probe_F and Sre1probe_R listed and labeled with phosphorus32-deoxycytidine triphosphate ([32P]-dCTP). The membrane was hybridized with the probe, exposed to a phosphor screen (PerkinElmer, Waltham, MA) overnight and scanned using a Packard cyclone phosphor imager (PerkinElmer).
Phosphor screen
Manufactured by PerkinElmer
Sourced in United States
The Phosphor screen is a lab equipment product that serves as a luminescent surface. It is designed to convert incident radiation, such as X-rays or charged particles, into visible light. The Phosphor screen is a key component in various imaging and detection applications within the scientific and medical fields.
Automatically generated - may contain errors
Lab products found in correlation
Packard cyclone phosphor imager, by PerkinElmer (2 mentions)
Typhoon trio variable mode imager, by GE Healthcare (2 mentions)
Optiquant software, by PerkinElmer (2 mentions)
Easytag express35s protein labelling mix, by PerkinElmer (1 mentions)
Cysteine, by PerkinElmer (1 mentions)
Endo hf, by New England Biolabs (1 mentions)
Ribopure yeast kit, by Thermo Fisher Scientific (1 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Cyclone storage phosphor system, by PerkinElmer (1 mentions)
Phosphor imaging screens, by PerkinElmer (1 mentions)
Nanozoomer, by Hamamatsu Photonics (1 mentions)
Cyclone plus imager, by PerkinElmer (1 mentions)
Cyclone plus system, by PerkinElmer (1 mentions)
2480 wizard2 automatic gamma counter, by PerkinElmer (1 mentions)
Cyclone phosphor imager system, by PerkinElmer (1 mentions)
Nupage gel, by Thermo Fisher Scientific (1 mentions)
8 protocols using phosphor screen
1
Generating sre1 Mutant by Biolistic Transformation
2
Pulse-Chase Analysis of Tapasin and TAPBPR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were starved for 30 min in methionine-free, cysteine-free RPMI medium, labelled using EasyTag Express [35S]-protein labelling mix (PerkinElmer) for 20 min, then chased in medium supplemented with 3 mM unlabelled l -methoinine (Sigma) and 60 μM unlabelled cysteine (Sigma) all at 37 °C. Samples taken at 0, 20, 45 and 90 min post-pulse, were subsequently washed in cold PBS, and lysed in 1% digitonin/TBS at 4 °C. Immunoprecipitation of tapasin and TAPBPR were performed as described above. Denatured samples were separated using SDS-PAGE. Gels were subsequently fixed in 12% acetic acid, 40% methanol and dried. Images were obtained using a phosphor screen (Perkin-Elmer) and on film. PhosphorImager analysis was performed using Typhoon Trio variable mode imager (GE Healthcare) together with ImageQuantTL software.
3
Metabolic Labeling and Immunoprecipitation of TAPBPR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Harvested cells were washed in PBS then incubated in cysteine/methionine-free RPMI-1640 supplemented with 5% dialysed fetal calf serum, 2 mm glutamine and 20 mm HEPES for 30 min at 37°. Cells were then labelled with [35S]methionine/cysteine Promix (Amersham Pharmacia) for 10 min at 37° and subsequently chased for 0–6 hr in RPMI containing 10% fetal calf serum supplemented with 3 mm unlabelled methionine and 60 μm unlabelled cysteine. After lysis in 1% digitoin TBS, TAPBPR was immunoprecipitated from pre-cleared post-nuclear lysates using the TAPBPR-specific mAb PeTe4 and protein A–sepharose. After extensive washing in 0·1% digitonin TBS, samples were heated at 80° for 10 min in sample buffer with 100 nm β-mercaptoethanol followed by digestion with 1000 U of endoglycosidase Hf (Endo Hf; NEB, Ipswich, MA) for 1 hr at 37°. After separation by SDS–PAGE, gels were fixed in 40% methanol, 12% acetic acid, dried and images were obtained on a phosphor screen (Perkin-Elmer, Waltham, MA). PhorphorImager analysis was performed using a Typhoon Trio variable mode imager (GE Healthcare) with imagequanttl software.
4
Iron Responsive Gene Expression Analysis
5
Ex vivo Autoradiography of Tumor Sections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tumors, spleen or mesenteric lymph nodes were fixed in formalin overnight, followed by paraffin embedding. Four µm sections were subsequently exposed overnight to a phosphor screen (PerkinElmer) in an X-ray cassette. Signal was detected with a Cyclone Storage Phosphor System (PerkinElmer). Slides used for ex vivo autoradiography were deparaffinized then stained with H&E and digitalized with NanoZoomer and NDP software (Hamamatsu). Subsequent slides were stained for GPC3 (tumor only) and CD3ε (online supplementary additional methods ).
For ex vivo tissue, autoradiography quantification of tumor sections, regions of interest (ROIs) were drawn for tumor cells and stromal regions based on H&E. ROIs were exported to ImageJ (National Institutes of Health, USA), rescaled for ex vivo autoradiography and ROIs were measured.
For tumor lysate and plasma analysis, samples were heated for 10 min at 70°C and 40 µg protein of tumor lysates or mouse plasma from three mice, tracer alone as positive control were loaded on mini-PROTEAN TGX Precast Gels (Bio-Rad). Gels were exposed overnight to phosphor imaging screens (PerkinElmer) in X-ray cassettes and analyzed using a Cyclone Storage Phosphor System (PerkinElmer).
For ex vivo tissue, autoradiography quantification of tumor sections, regions of interest (ROIs) were drawn for tumor cells and stromal regions based on H&E. ROIs were exported to ImageJ (National Institutes of Health, USA), rescaled for ex vivo autoradiography and ROIs were measured.
For tumor lysate and plasma analysis, samples were heated for 10 min at 70°C and 40 µg protein of tumor lysates or mouse plasma from three mice, tracer alone as positive control were loaded on mini-PROTEAN TGX Precast Gels (Bio-Rad). Gels were exposed overnight to phosphor imaging screens (PerkinElmer) in X-ray cassettes and analyzed using a Cyclone Storage Phosphor System (PerkinElmer).
6
Radiological Imaging of Aortic Aneurysm
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following a 90-minute delay for biodistribution and gamma counting, spleen, control aorta, and 14-day AngII AAA whole-organ samples were placed on a phosphor screen (PerkinElmer) covered with clear cellophane wrap. After overnight exposure, the imaging plates were scanned using the Cyclone Plus imager (PerkinElmer) at a resolution of 300 DPI. Images were processed using OptiQuant software (PerkinElmer).
7
Radiolabeling of Fe3O4@Al(OH)3 Nanoparticles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To evaluate the time course of radiolabeling, 45 µL of Fe3O4@Al(OH)3 NPs containing 60 µg of Fe were incubated with 5–30 MBq [18F]NaF (5–30 µL) while shaking. Two, five, and 10 min after labeling, 2 µL samples were taken and blotted on instant thin layer chromatography (iTLC) papers impregnated with silica gel (iTLC-SG papers; Varian, Diegem, Belgium). The papers were developed in an elution chamber using NaCl 0.9% as the mobile phase. The read-out was performed using a 2480 Wizard2 Automatic Gamma Counter (20 s protocol; PerkinElmer, Waltham, MA, USA) after splitting the papers into two equal halves representing the unbound and bound radiotracer. In addition, autoradiography was performed using phosphor screens (Perkin Elmer) in standard film cassettes. The screens were removed from the cassettes after a five-minute exposure and were scanned immediately at 300 dpi resolution using a Cyclone Plus System (Perkin Elmer). Images were analyzed using the manufactures’ Optiquant software (version 5; Perkin Elmer).
For further in vitro and in vivo experiment, NPs are labeled with [18F]NaF for ten minutes in Milli-Q water. Afterward, they are centrifuged for 20 min at 4000 rpm and resuspended in the media of choice for further application.
For further in vitro and in vivo experiment, NPs are labeled with [18F]NaF for ten minutes in Milli-Q water. Afterward, they are centrifuged for 20 min at 4000 rpm and resuspended in the media of choice for further application.
8
Competitive Radiolabeling of Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A non-His-tagged protein (C2Ac [12 (link)]) was allowed to compete for radiolabel with a His-tagged protein (α-CD33 scFv) by incubating an equimolar mixture of both proteins (55 μM:55 μM) in a one-pot radiolabelling reaction using the IsoLink kit method 2 described above. As controls, the proteins were radiolabelled separately using 55 μM concentrations. The reactions were allowed to incubate for 1 h at 37°C, after which 5 μL samples were taken from each reaction mixture and loaded onto an SDS-PAGE gel (12% NuPage gel, Invitrogen, Paisley, PA, UK). After the gel was run, a reference lane with molecular weight markers was spotted with radioactivity at the 15- and 30-kDa bands. After protein separation via electrophoresis, phosphor screens (Perkin Elmer, Shelton, CT, USA) were exposed for 10 s, followed by imaging using a Cyclone® phosphorimager system (Perkin Elmer). The gel was analysed using ImageJ software (NIH, Bethesda, MD, USA), and the fraction of total radioactivity bound to each of C2Ac and α-CD33 scFv was determined via densitometry of the protein bands.
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!