Protein expression was detected using western blot analysis as previously described (17 (link)). Lysates were prepared using log phase trophozoites. Trophozoites were iced, pelleted and washed in ice cold PBS, then resuspended in NETN lysis buffer (100 mM NaCl, 20 mM Tris pH 8.0, 1 mM EDTA, 0.2% NP-40) containing 50 μM E-64 and 1 x HALT inhibitor cocktail, incubated 10 min on ice, followed by centrifugation at 14,000 g for 10 min. The remaining pellets were dissolved in 8 M Urea solution. Lysates were applied to a 10–12% SDS-PAGE gel and transferred onto a nitrocellulose membrane. The membrane was blocked with 5% milk in PBS-T (PBS containing 0.1% Tween-20) and incubated with antibodies against Myc (mouse) (1:1000 dilution; Cell Signaling Technology, USA), EhROM1 (rat) (1:20 dilution; custom made by Harlan, USA), EhROM3 (rabbit) (1:500 dilution; custom made by Pierce Biotechnology, USA), and Actin (mouse) (MP Biomedicals 1:1000) followed by incubation with secondary mouse or rat horseradish peroxidase (HRP)-conjugated antibody (1:1,000 dilution; Cell Signaling Technology or Santa Cruz Biotechnology, USA) and developed using enhanced chemiluminescence (ECL Prime) (GE Healthcare, USA). Blots were either scanned on a Kodak Image Station 4000R or imaged on film and developed using a Kodak X-OMAT 2000 processor.
X omat 2000 processor
Manufactured by Kodak
Sourced in United States
The X-OMAT 2000 Processor is a piece of lab equipment designed for the automated processing of x-ray film. It is capable of developing, fixing, washing, and drying x-ray film in a controlled environment to produce high-quality images for diagnostic or other purposes.
Automatically generated - may contain errors
Lab products found in correlation
Dneasy blood tissue kit, by Qiagen (2 mentions)
Image station 4000r, by Kodak (1 mentions)
Ecl prime, by GE Healthcare (1 mentions)
1 kb dna ladder, by New England Biolabs (1 mentions)
Dig high prime dna labeling and detection starter kit 2, by Roche (1 mentions)
Mxg film, by Kodak (1 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Phosphatase inhibitor, by Merck Group (1 mentions)
Enhanced chemiluminescent reagent, by Thermo Fisher Scientific (1 mentions)
Restore western blot stripping buffer, by Thermo Fisher Scientific (1 mentions)
Positively charged nylon membrane, by Roche (1 mentions)
X ray film, by Carestream (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Hinfi and rsai restriction enzymes, by New England Biolabs (1 mentions)
Chef dr 2 equipment, by Bio-Rad (1 mentions)
Telotaggg telomere length assay kit, by Roche (1 mentions)
X ray film, by Kodak (1 mentions)
Positively charged nylon membrane, by GE Healthcare (1 mentions)
Dig high prime dna labelling and detection starter kit 2, by Roche (1 mentions)
12 protocols using x omat 2000 processor
1
Western Blot Analysis of Protein Expression
2
DNA Purification and Southern Blotting
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Genomic DNA was purified using DNeasy Blood & Tissue Kit (Qiagen, GmbH). 15 μg of genomic DNA was subjected to restriction digestion using 50 U of the respective enzyme in 200 μl overnight at 37°C. DNA was ethanol precipitated and dissolved in 20 μl TE buffer (pH 8.0). Targeting vectors were linearized with single cutter restriction enzyme and diluted to 107, 108, 109 copies per μl. Digested genomic DNA samples were resolved overnight on a 1% agarose gel in 1× TAE (Tris-Acetate-Boric acid) buffer, with 1 kb DNA ladder (New England Biolabs) and 1 μl of positive control samples. Southern blotting employing the respective probes, as indicated, was performed using the DIG-High Prime DNA Labeling and Detection Starter Kit II (Roche) as per the manufacturers’ protocol. The probe-target hybrids on the blots were detected by chemiluminescent assay followed by exposure to an X-ray film (Kodak MXG film, Kodak) and developed on a Kodak X-OMAT 2000 Processor.
3
Protein Extraction and Western Blot Analysis
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Whole-cell lysates were made by subjecting AS-10-treated Panc-1 cells to RIPA lysis buffer (Thermo Scientific, Waltham, MA, USA) containing cocktails of protease inhibitors (Roche, Little Falls, NJ, USA) and phosphatase inhibitors (Sigma-Aldrich, USA) as described before [22 (link)]. Lysates were spun at 15,000 rpm for 10 min. The resulting supernatant was stored at −80 °C until use. NuPAGE gel 4–12% (Life Technologies, Carlsbad, CA, USA) was used to resolve the lysates (30 μg of protein per sample), followed by electro-transfer to the PVDF membrane. After the transfer, the membrane was blocked with 5% non-fat milk and probed with different antibodies. Protein of interest were detected by using an enhanced chemiluminescent reagent (Life technologies, Carlsbad, CA, USA) and X-ray films were developed using a Kodak X-OMAT 2000 processor. Blots were stripped using Restore Western blot stripping buffer (Thermo Scientific, USA) and used for loading control or other protein detection to save time and samples.
4
Assaying PrimPol Priming Activity
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Single-stranded M13mp18 DNA (5 nM) was used as template to assess priming activity of PrimPol (400 nM) in the presence of indicated dNTPs (10 µM) and 16 nM [α-32P]dGTP (250 µCi; 3000 Ci/mmol). Reaction mixtures (20 µL) in Buffer R, were incubated 30 min at 30 °C then stopped by adding 8 μL of formamide loading buffer, and loaded onto 8 M urea-containing 20% polyacrylamide sequencing gels (60 cm) and run 2 h at 50 W. After electrophoresis, products were detected by autoradiography using AGFA CP-BU NEW Healthcare NV Medical X-RAY films blue (Ref. EWPKK, Mortsel, Belgium) and developed by a Kodak X-OMAT 2000 Processor (Rochester, NY, USA).
5
Measuring Telomere Length via Restriction Fragment Analysis
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Telomere restriction fragments were analyzed using the TeloTAGGG Telomere Length Assay kit (Roche). Briefly, cells were harvested by trypsinization, washed in PBS and collected by centrifugation at 400 g for 4 min. Genomic DNA was isolated using DNeasy Blood and Tissue Kit (Qiagen), digested with HinfI and RsaI restriction enzymes (New England Biolabs) and separated by gel electrophoresis either on 0.8% agarose gels at 50V overnight in 1X TBE buffer or (to resolve elongated telomeres at later time points) on 1% megabase agarose gels (Bio-Rad) using a CHEF DRII equipment (Bio-Rad) under the following conditions: 120° field angle, 5 to 30 s switch times, 5 V/cm and 14°C for 14 hr in 1X TAE. Following the resolution of DNA fragments, DNA was transferred to a positively charged nylon membrane (Roche) by Southern blotting and hybridized with a digoxigenin-labelled telomeric probe. Membranes were exposed to X-ray film (Carestream) and developed in X-OMAT 2000 Processor (Kodak). Mean telomere lengths were calculated as described in Kimura et al. (2010) (link).
6
Primer Extension Assay for PrimPol
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Full extension of a specific sequence substrate used a primer: 5′-CTGCAGCTGATGCGC-3′ and template: 5′ -GTACCCGGGGATCCGTACGGCGCATCAGCTGCAG-3′ (in a 1:2 ratio). Reaction mixtures (in 20 µL) contained Buffer R [50 mM Tris–HCl pH 7.5, 40 mM NaCl, 2.5% (w/v) glycerol, 1 mM DTT, 0.1 mg/mL BSA, 1 mM MnCl2], 2.5 nM [γ-32P]-labeled primer:template, 200 nM purified PrimPol and dNTPs (1, 10 100 µM). Reactions were incubated during 30 min at 30 °C, and stopped by adding 8 μL of formamide loading buffer, then loaded onto 8 M urea-containing 20% polyacrylamide sequencing gels of 30 cm long and run 2 h at 30 W. Following denaturing electrophoresis, primer extension was detected by autoradiography using AGFA CP-BU NEW Healthcare NV Medical X-RAY films blue (Ref. EWPKK, Mortsel, Belgium) and developed by a Kodak X-OMAT 2000 Processor (Rochester, NY, USA).
7
Southern Blot Analysis of Genomic DNA
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Genomic DNA was isolated from parental hESCs and clones using the DNeasy Blood & Tissue Kit (Qiagen). Approximately 20 μg of each DNA was digested with a suitable restriction enzyme (New England Biolabs) overnight at 37 °C. Genomic DNA fragments were separated by electrophoresis on a 0.8% agarose gel in 1x TAE (Tris-Acetate-Boric acid) buffer, with 1 kb DNA marker ladder (New England Biolabs) and transferred onto a positively charged nylon membrane (GE Healthcare) via capillary transfer method. The DNA on the membrane was UV crosslinked and the membrane was probed at 48 °C with PCR-amplified DIG-labelled NeoR probe using the DIG-High Prime DNA Labelling and Detection Starter Kit II (Roche) as per the manufacturers’ protocol. The probe-target hybrids on the blot were detected by an AP-conjugated DIG-Antibody (Roche) using CSPD (Roche) as a substrate for chemiluminescence. The blots were exposed to X-Ray film (Kodak) and developed on a Kodak X-OMAT 2000 Processor.
8
Western Blot Protein Analysis Protocol
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Cell monolayers were washed three times with ice-cold phosphate-buffered saline (PBS) and lysed at 4°C in 500 μl of lysis buffer (10 mM HEPES pH 8.0, 100 mM NaCl, 1 mM MgCl2 and 1% Triton X-100) containing 10 μg/ml aprotinin, 0.1 mM PMSF and 10 μg/ml leupeptin. Lysates were incubated on ice for 30 minutes and centrifuged at 10,000 g for 15 minutes at 4°C. Samples, containing the same amount of protein, were electrophoresed through 10% SDS-PAGE and electroblotted onto a nitrocellulose membrane (Schleicher and Schuell BioScience, Keene, NH, USA). Membranes were blocked with 5% skim milk and 0.1% Tween-20 in Tris-buffered saline (TBS) for 1 hour at room temperature (RT) and incubated overnight with the primary antibody. The membranes were then washed three times in 0.1% Tween-20 in TBS and incubated with the appropriate secondary antibody for 1 hour at RT. After washing, membranes were reacted with ECL detection reagents (Santa Cruz Biotechnology Inc., CA, USA) and analyzed by luminescent image analyzer (X-OMAT 2000 Processor, Kodak, Rochester, NY, USA).
9
Protein Expression Analysis of Mammary Tissues
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Mouse mammary tumors and normal mammary tissue were minced, digested, and processed to separate mammary organoids as previously described. Cell aggregates were resuspended in RIPA lysis buffer (10mM Tris-HCl, pH 8.0, 1mM EDTA, 0.5mM EGTA, 1% Triton X-100, 0.1% Sodium Deoxycholate, 0.1% SDS, 140mM NaCl) containing protease and phosphatase inhibitors, mechanically disaggregated using a Polytron ® tissue homogenizer, and lysed for 2 h under rotation at 4°C. Cell lysates were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were then blocked for 1 h in TBS-Tween with 5% BSA and incubated overnight at 4°C with primary antibodies diluted in blocking buffer. The primary antibodies used included anti-phospho-AKT S473 (#4060), phospho-FoxO1 (Thr24)/FoxO3a (Thr32) (#9464), phospho-MEK1/2 (Ser217/221) (#9121), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (#4370), phospho-S6 Ribosomal Protein (Ser240/244) (#2217), and β-Actin (#4967) from Cell Signaling, and anti-HA from Sigma. Primary antibodies were used at 1:1000 dilution, except β-Actin at 1:20,000. The next day, membranes were washed twice with TBS-Tween and incubated for 45 min at RT with secondary antibody (1:30,000, Jackson Immunoresearch). After at least four washes, antibody binding was detected using standard chemiluminescence with a Kodak X-OMAT 2000 Processor.
10
Primase Assay for Measuring PrimPol Activity
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Primase assays were carried out using the following unlabeled ssDNA oligonucleotides as templates: 3′-(T)20GTC C(T)36–5′ or 3′-(T)20GTC AGACAGCA(T)29–5′. The reaction mixture (20 µL) in Buffer R contained 1 µM ssDNA template, 400 nM PrimPol, 16 nM [γ-32P]ATP or [α-32P]dGTP and indicated dNTPs at 10 µM. Dimer synthesis experiment was measured using ATP as a 5′nucleotide (1, 10, 100 µM). Pre-made 3PAGT primer (10 µM) (synthesized by IDT, Coralville, IA, USA) was used to measure the elongation capacity of PrimPol variants. After an incubation time of 30 min at 30 °C, reactions were stopped adding 8 μL of formamide loading buffer. Synthesized primers were resolved in a 8 M urea-containing 20% polyacrylamide sequencing gels (60 cm) and run 2 h at 50 W. After electrophoresis, products were detected by autoradiography using AGFA CP-BU NEW Healthcare NV Medical X-RAY films blue (Ref. EWPKK, Mortsel, Belgium) and developed by a Kodak X-OMAT 2000 Processor (Rochester, NY, USA)..
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