Total RNA was isolated from fibroblasts using QIAzol Lysis Reagent (Qiagen SpA, Milan, Italy), and reverse transcription was performed as described in (Ferrera et al., 2021 (link); Negri et al., 2021 (link)). Reverse transcription was always performed in the presence (positive) or in the absence (negative control) of the reverse transcriptase enzyme (not shown), as shown elsewhere (Zuccolo et al., 2019 (link); Zuccolini et al., 2022 (link)). cDNA amplification was performed using KAPA SYBR FAST qPCR Master Mix (KAPA BIOSYSTEMS, United States), and the primers used for amplification are listed the Table 1 . The conditions were as follows: initial denaturation at 95°C for 5 min; 40 cycles of denaturation at 95°C for 10 s; annealing and extension at 60°C for 30 s, PCR products were separated on a 3% Nusieve® (2:1) gel agarose, stained with ethidium bromide, and acquired with the iBrightTM CL1000 Imaging System (Thermo Fisher Scientific Inc., United States). The molecular weight of the PCR products was compared with the DNA molecular weight marker VIII (Roche Molecular Biochemicals, Italy).
Ibrighttm cl1000 imaging system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The IBrightTM CL1000 Imaging System is a compact, versatile imager designed for biologists and life science researchers. It captures high-quality images of chemiluminescent and fluorescent samples, including Western blots, ELISA plates, and other gel-based applications. The system features a sensitive CCD camera, a motorized sample stage, and various filter options to optimize image acquisition.
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Lab products found in correlation
Ab16901, by Abcam (2 mentions)
Rabbit anti gapdh, by Cell Signaling Technology (2 mentions)
Ecl western blotting substrate, by Thermo Fisher Scientific (2 mentions)
Mouse anti crispr cas9 antibody, by Abcam (2 mentions)
Mouse anti gapdh antibody, by ABclonal (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Dna molecular weight marker 8, by Roche (1 mentions)
Kapa sybr fast qpcr master mix, by Roche (1 mentions)
Ab109268, by Abcam (1 mentions)
Ab53290, by Abcam (1 mentions)
Agarose gel, by Lonza (1 mentions)
Ab40660, by Abcam (1 mentions)
Ab179461, by Abcam (1 mentions)
Rabbit anti vimentin, by Cell Signaling Technology (1 mentions)
Rabbit anti β catenin, by Cell Signaling Technology (1 mentions)
Rabbit anti akt, by Cell Signaling Technology (1 mentions)
Mouse anti e cadherin, by Cell Signaling Technology (1 mentions)
7 protocols using ibrighttm cl1000 imaging system
1
Total RNA Isolation and Reverse Transcription
2
Western Blot Analysis of Signaling Pathways
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ZR75 cells were seeded in 100 mm petri dishes at a density of 2,000,000 cells/dish. Cells were treated with TZ, BMS-202, or a combination of both for 48 hours. Cell lysates were collected, and 30 μg of proteins were resolved on 10% polyacrylamide SDS PAGE gels and then transferred onto PVDF membranes. Membranes were probed with the following primary antibodies: anti-rabbit Akt (CST: 9272S), anti-rabbit phospho-Akt (Ser473) (CST: 4060S), anti-rabbit mTOR (CST: 2983S), anti-rabbit phospho mTOR (S2448) (Abcam: ab109268), anti-mouse ErbB2 (Abcam: ab16901), anti-rabbit phospho ErbB2 (Abcam: ab53290), and anti-rabbit vimentin (CST: 46173S). Anti-rabbit GAPDH (Cell Signaling: 8480S) was used to ensure equal loading of protein samples. Blots were incubated with ECL Western blotting substrate (Pierce Biotechnology, Rockford, IL, USA) and chemiluminescence was recorded using the iBrightTM CL1000 imaging system (Thermo Fisher Scientific, Wal-tham, MA, USA). Quantification was done using ImageJ software.
3
Analyzing Genetic Diversity via flaA-RFLP
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Genetic diversity was first analyzed by flaA-restriction fragment length polymorphism (RFLP) using 25-μl PCR reactions (Harrington et al., 2003 (link); Wieczorek and Osek, 2008 ). The flaA amplicon (1.7 kb) was digested for 6 h at 37°C using HpyF3I restriction enzyme (Thermo Scientific, Waltham, MA, United States), and the fragments were separated using 2.5% agarose gel (Lonza Inc., Rockland, ME, United States) in Tris–acetate–EDTA buffer at 90 V for 90 min. The bands were photographed with iBrightTM CL1000 Imaging System (Thermo Fisher Scientific, Seoul, Republic of Korea). The dyne 100-bp and 1-kb DNA ladders (Dyne bio, Seongnam-si, Republic of Korea) were used as standards for molecular size determination.
4
Western Blot Analysis of Protein Expression
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Alterations in the protein expression levels were analyzed by Western blotting as previously described by our group [42 (link)]. In brief, SKBR3 and ZR75 cells (2 × 106 cells) were seeded and treated with DA and BMS-202, individually and in combination for 48 h. Cell lysates were collected, and equal amounts of protein (30 µg) were resolved on 10% SDS PAGE gels and electroblotted onto PVDF membranes, then probed with the following primary antibodies: anti-rabbit Src family (phospho Y418) (Abcam: ab40660), anti-mouse ErbB2 (Abcam: ab16901), anti-rabbit phosphorylated ErbB2 (Abcam: ab53290), anti-mouse E-cadherin (Cell Signaling: 14,472 S), anti-rabbit vimentin (Cell Signaling: 46,173 S), anti-rabbit β-catenin (Cell Signaling: 8480 S), anti-rabbit phosphorylated β-catenin (Cell Signaling: 4176 S), anti-rabbit AKT (Cell Signaling: 9272 S), anti-rabbit phosphorylated AKT (Cell Signaling: 4060 S), anti-rabbit JNK1/2/3 (Abcam: ab179461). Anti-rabbit GAPDH (Cell Signaling: 8480 S) was used to ensure equal loading of protein samples.
Immunoreactivity was analyzed using the ECL Western blotting substrate (Pierce Biotechnology, Rockford, IL, USA), as described by the manufacturer, and blots were imaged using the iBrightTM CL1000 imaging system (Thermo Fisher Scientific, Waltham, MA, USA). Quantification was done using ImageJ software as previously described by our group [43 ].
Immunoreactivity was analyzed using the ECL Western blotting substrate (Pierce Biotechnology, Rockford, IL, USA), as described by the manufacturer, and blots were imaged using the iBrightTM CL1000 imaging system (Thermo Fisher Scientific, Waltham, MA, USA). Quantification was done using ImageJ software as previously described by our group [43 ].
5
Analyzing PDE6B Expression in Mouse Retina
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Western blot analyses were performed on retinal lysates from mouse eye. The mouse eye retina was dissected and placed in a microcentrifuge tube containing radioimmunoprecipitation assay (RIPA) buffer (50 μL RIPA/retina). To determine the effect of bystander editing on PDE6B expression, three randomized retinas of two mice in the WT, untreated, AAV8-W599R-PDE6B, and AAV8-WT-PDE6B groups were homogenized (n = 3 retinas/group). To explore whether the corrected Pde6b gene could be translated into PDE6B protein, three randomized retinas of two mice in the WT, untreated, or untargeted group were homogenized (n = 3 retinas/group). Six retinas of three 6E9-treated mice were randomly divided into two portions for homogenization (n = 3 retinas/portion).The total protein concentration was determined by BCA protein assay (Thermo Fisher Scientific; Waltham, MA). PDE6B protein was detected by Rabbit anti-PDE6B antibody (1:500; Thermo Scientific, PA1-722). Mouse anti-GAPDH antibody (1:10,000; ABclonal; Cat# AC002) was used to detect GAPDH. Blots were imaged and analyzed by iBrightTM CL1000 imaging systems (Thermo Scientific).
6
CRISPR-Cas9 Protein Detection
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Western blot analyses were performed on cell lysates. SpCas9 protein was detected by Mouse anti-CRISPR-Cas9 antibody (1:1000 dilution, Abcam, Cat# 191468). Mouse anti-GAPDH antibody (1:10000 dilution, ABclonal, Cat# AC002) was used to detect GAPDH. Blots were imaged and analyzed by iBrightTM CL1000 imaging systems (Thermo FisherScientific, Invitrogen™).
7
Quantifying CRISPR-Cas9 Protein Expression
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Western blot analyses were performed on cell lysates. SpCas9 protein was detected by Mouse anti-CRISPR-Cas9 antibody (1:1000 dilution, Abcam, Cat# 191468). Mouse anti-GAPDH antibody (1:10000 dilution, ABclonal, Cat# AC002) was used to detect GAPDH.
Blots were imaged and analyzed by iBrightTM CL1000 imaging systems (Thermo FisherScientific, InvitrogenTM).
Blots were imaged and analyzed by iBrightTM CL1000 imaging systems (Thermo FisherScientific, InvitrogenTM).
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