Western blotting was performed as previously described [49 (link)]. Briefly, equal amounts of lysates were resolved with SDS-PAGE and were transferred onto PVDF membranes. Membranes were incubated with primary antibodies followed by horseradish peroxidase (HRP)-conjugated secondary antibodies. Signal amplification and detection were achieved by exposing the membrane to enhanced chemiluminescence reagent (GE Healthcare, Buckinghamshire, UK), followed by visualization using the Storm imaging system (Amersham Biosciences, Piscataway, NJ, USA). The following primary antibodies were used: γ-H2AX (1/1000; Cell Signaling Technology, Beverly, MA, USA), Chk2 (1/500; Cell Signaling Technology), Phospho-Chk2 (Thr68) (1/1000; Cell Signaling Technology), XRCC2 (1:1500; Abcam), Caspase-9 (1/500; Proteintech, Chicago, IL, USA), Caspase-3 (1/500; Proteintech), PARP (1/500; Proteintech), and BCL-2 (1/500; Santa Cruz Biotechnology). Detection of GADPH (1/10000; Cell Signaling Technology) was used as a loading control. Bound antibodies were visualized with peroxidase-linked secondary antibodies (anti-rabbit antibody: 1/10000; Cell Signaling Technology and anti-mouse antibody: 1/5000; Sigma-Aldrich, St. Louis, MO, USA).
Storm imaging system
Manufactured by Cytiva
Sourced in United Kingdom
The Storm imaging system is a high-performance protein gel imaging solution designed for life science laboratories. It provides efficient and accurate detection of protein bands and other molecular targets in gel-based electrophoresis applications.
Automatically generated - may contain errors
Lab products found in correlation
Ready to go dna labelling system, by GE Healthcare (2 mentions)
Hybond n, by Cytiva (2 mentions)
Goat anti mouse antibody, by Santa Cruz Biotechnology (2 mentions)
Caspase 9, by Proteintech (1 mentions)
Xrcc2, by Abcam (1 mentions)
Enhanced chemiluminescence reagent, by GE Healthcare (1 mentions)
γh2ax, by Cell Signaling Technology (1 mentions)
Phospho chk2 thr68, by Cell Signaling Technology (1 mentions)
Anti rabbit antibody, by Cell Signaling Technology (1 mentions)
Anti mouse antibody, by Merck Group (1 mentions)
Gadph, by Cell Signaling Technology (1 mentions)
Caspase 3, by Proteintech (1 mentions)
Bcl 2, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Ecl plus reagent, by Cytiva (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
β actin, by Abcam (1 mentions)
Imagequant tl, by Cytiva (1 mentions)
Ecl substrate, by Cytiva (1 mentions)
Anti p53, by Cell Signaling Technology (1 mentions)
8 protocols using storm imaging system
1
Western Blot Analysis of DNA Damage Response
2
Western Blot Analysis of CDK6 and β-Actin Proteins
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The protein samples were heated at 95°C for 10 min with the sample buffer (250 mM Tris-HCl, 4% sodium dodecyl sulfate, 2% β-mercaptoethanol, 10% glycerol and 0.003% bromophenol blue) and separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The protein samples were then transferred onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA), which were blocked with 5% skimmed dry milk in Tris-buffered saline (TBS) with 0.1 % Tween 20 (TBS-T) for 1 h. The membranes were then incubated overnight at 4°C with primary antibody diluted in 0.3% bovine serum albumin (BSA)-TBS-T. The membranes were incubated with the primary antibodies (CDK6, 1:1,000 and β-actin, 1:400; Abcam, Cambridge, UK) at 4°C for 12 h, and then with the horseradish peroxidase-linked secondary antibodies (1:1,000, goat anti-mouse monoclonal CD151 and goat anti-rabbit monoclonal β-actin; Abcam) at 37° for 1 h. The membranes were incubated with ECL Plus reagent (Amersham Biosciences, Uppsala, Sweden) and scanned using the Storm imaging system (Amersham Biosciences). Immunoreactive products were quantified using Quantity One software (Bio-Rad, Hercules, CA, USA) by determining the optical density of the protein bands.
3
In Vitro Radiolabeling of PhaC
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In a final volume of 22 μL, PhaC
(4 μM) was first reacted at room temperature for 1 min with
0 or 10 equiv of sTCoA and then chased by 5 or 50 equiv of [1-14C]HBCoA (88000 cpm total per reaction). After 10 s, 10 μL
aliquots were withdrawn and quenched in 10 μL of Laemmli buffer
(without reducing agent), and the samples were not boiled. PhaCCc standards and the samples (20 μL) were immediately
loaded onto two 10% SDS–PAGE gels and run at 150 V on ice for
1 h. One gel was stained for 15 min in fresh Coomassie stain and then
destained for 15 min in fast destain. The second gel was rinsed for
3 × 5 min in ddH2O, immediately dried, and then exposed
to a low-energy phosphor screen (Molecular Dynamics) for 12 h. The
phosphor screen was scanned using the Storm Imaging System and analyzed
using ImageQuant TL (Amersham Biosciences).
(4 μM) was first reacted at room temperature for 1 min with
0 or 10 equiv of sTCoA and then chased by 5 or 50 equiv of [1-14C]HBCoA (88000 cpm total per reaction). After 10 s, 10 μL
aliquots were withdrawn and quenched in 10 μL of Laemmli buffer
(without reducing agent), and the samples were not boiled. PhaCCc standards and the samples (20 μL) were immediately
loaded onto two 10% SDS–PAGE gels and run at 150 V on ice for
1 h. One gel was stained for 15 min in fresh Coomassie stain and then
destained for 15 min in fast destain. The second gel was rinsed for
3 × 5 min in ddH2O, immediately dried, and then exposed
to a low-energy phosphor screen (Molecular Dynamics) for 12 h. The
phosphor screen was scanned using the Storm Imaging System and analyzed
using ImageQuant TL (Amersham Biosciences).
4
Intestinal Crypt and Villi Protein Analysis
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Mechanically separated crypts and villi of small intestine [46 (link)], were homogenized and lysed using RIPA buffer (25mM Tris•HCl pH 7.6, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, Thermo Scientific, #89901) supplemented with protease inhibitor cocktail (Roche). Supernatants were collected after centrifugation at 14,000 r.p.m for 1 hour. Protein concentration was determined by the BCA method (Pierce, Thermo Scientific). Proteins were subjected to SDS−PAGE and immunoblot analysis. Blots were probed sequentially with primary and secondary antibodies at the following dilutions: anti-p53 at 1:1000 (1C12, Cell Signaling), anti-Actin at 1:5000 (Sigma-Aldrich Corporation, St. Louis, USA). Secondary HRP-conjugated anti-mouse and anti-rabbit were used at 1:10 000 (GE Healthcare, Chalfont St Giles, UK). Proteins were detected by incubation with ECL substrate (Amersham Bioscience, Piscataway, USA) for 5 min and chemiluminescence was visualized by STORM imaging system (Amersham, Pleasanton, USA).
5
HPV16 Transcriptional Analysis in Cell Lines
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Total RNAs from cells (C33a, SiHa) transfected with pIRES-16E1/E2/E4, pIRES-16E1/E2 or pIRES (empty plasmid) were extracted using RNAeasy mini Kit (Qiagen). RNA samples were electrophoresed on a 1.2% formaldehyde agarose gel. Following electrophoresis, RNAs were transferred to a nylon membrane (Hybond-N; Amersham, Piscatway, NJ) and hybridized to [α-32P]dCTP-radiolabeled DNA probe for E1E2E4 DNA fragment of HPV16 prepared with Ready-to-Go DNA labelling system (GE Healthcare Life science). Radioactive bands were detected using a Storm imaging system (Amersham). APOT assay using RNA sample of NIKS/HPV16 was carried out following the protocol described previously [43 (link)].
6
Western Blot for XRCC2 Protein
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Proteins from cell lysates were prepared, separated on SDS–PAGE, and transferred to polyvinyl difluoride (PVDF) membranes according to the manufacturers’ instructions. Anti-human XRCC2 mouse monoclonal antibody (1:1500; Abcam) and anti-β-actin mouse monoclonal antibody (1:1000; Sigma–Aldrich) were used as primary antibodies for detecting specific proteins. Goat anti-mouse antibody (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as the secondary antibody. Signal amplification and detection were achieved by exposing the membrane to electrochemiluminescence reagent (GE Healthcare, Buckinghamshire, UK), followed by visualization using the Storm imaging system (Amersham Biosciences, Piscataway, NJ, USA).
7
Western Blot Analysis of DNA Damage Markers
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Proteins from cell lysates were prepared, separated on SDS-PAGE, and transferred to PVDF membranes, according to the manufacturer’s instructions. Anti-human γH2AX mouse monoclonal antibody (1:1,000; CST, Boston, MA, USA), anti-human PARP1 mouse monoclonal antibody (1:1,000; Abcam, Cambridge, UK), and anti-β-tubulin mouse monoclonal antibody (1:1,500; Sigma-Aldrich, St Louis, MO, USA) were used as primary antibodies. Goat anti-mouse antibody (1:2,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as a secondary antibody. Signal amplification and detection were achieved by exposing the membrane to ECL reagent (GE Healthcare), followed by visualization on the storm imaging system (Amersham Biosciences).
8
Genomic DNA Profiling of HPV16
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Total genomic DNAs were isolated using QIAamp DNA blood mini kit. Total genomic DNAs isolated from each population were digested with either BamHI or HindIII for at least 8h at 37°C and electrophoresed on a 0.8% agarose gel. Total genomic DNA isolated from untransfected NIKS cells was used as a negative control. Following electrophoresis, DNAs were transferred to a nylon membrane (Hybond-N; Amersham, Piscatway, NJ) and hybridized to [α-32P]dCTP-radiolabeled DNA probe for full-length HPV16 prepared with Ready-to-Go DNA labelling system (GE Healthcare Life science). Radioactive bands were detected using a Storm imaging system (Amersham).
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