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Chemidoc chemiluminescence system

Manufactured by Bio-Rad
Sourced in United States
About the product

The ChemiDoc™ chemiluminescence system is a laboratory instrument designed for the detection and analysis of chemiluminescent signals. It is capable of imaging and quantifying chemiluminescent samples, such as those used in Western blotting, ELISA, and other protein detection applications.

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13 protocols using chemidoc chemiluminescence system

1

Characterization of T. gondii Proteome

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Completely egressed tachyzoites of T. gondii were collected and lysed in ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology, China) supplemented with protease and phosphatase inhibitor cocktails (Bimake, USA), and they were incubated at 4 °C for 30 min to generate protein lysate. After 100 ℃ denaturation, protein lysate was subjected to polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The PVDF membrane was blocked with 5% (w/v) skimmed milk and then incubated with primary and secondary antibodies. Signals were visualized by a standard chemiluminescent HRP method with the ECL reagent (Fude, China), and detected using the ChemiDoc chemiluminescence system (Bio-Rad, Hercules, USA).
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2

In Vivo CRISPR-Cas9 Editing in Muscular Dystrophy Mice

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ITR-containing plasmids, AAV-2XgRNA (9 (link)), AAV-WT-SaCas9 (9 (link)) and AAV-AEGA-SaCas9 were generated in Dr Charles Gersbach‘s Lab (Duke University). Intact ITRs were verified by SmaI digest on all vectors before AAV production. AAV9 was produced by the Duke University Viral Vector Core and titers were measured by qPCR with a plasmid standard curve. Animal studies were conducted with strict adherence to the guidelines for the care and use of laboratory animals of the National Institutes of Health (NIH). All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Duke University. Neonatal 2-day-old (P2) mdx mice (Jackson Labs) were administered AAV by intravenous injection through the facial vein with 40ul AAV vector per mouse (7.2e11vg AAV-2XgRNA and 2.2e11vg AAV-WT-SaCas9 or AAV-AEGA-SaCas9. At 8 weeks post-injection, mice were euthanized and tibialis anterior muscle tissue was collected. Protein analysis with western blot and genomic DNA analysis with endpoint PCR were performed as described previously (9 (link)). In brief, protein lysate was loaded onto NuPAGE Bis-Tris gels (Invitrogen) with MOPS buffer (Invitrogen), transferred to nitrocellulose membranes, and blocked overnight. Blots were probed with anti-HA (abcam) for Cas9 detection, MANDYs8 (Sigma D8168) for dystrophin detection, and rabbit anti-GAPDH (Cell Signaling), followed by mouse or rabbit horseradish peroxidase-conjugated secondary antibodies (Santa Cruz) and visualized using Western-C ECL substrate (Biorad) on a ChemiDoc chemiluminescence system (Biorad). In brief, genomic DNA was isolated using the DNeasy kit (Qiagen) and endpoint PCR was performed with primers flanking the Cas9 cut sites (5′-TACACTAACACGCATATTTG, 5′-CATTGCATCCATGTCTGACT) to amplify a 1638 bp unedited region or the expected 467 bp deletion product. PCR products were electrophoresed in a 1% agarose gel and viewed on a BioRad GelDoc imager.
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3

Comprehensive Protein Analysis in Cells

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Total protein extraction from cells was performed using RIPA lysis buffer (P0013B, Beyotime, China), followed by determination of protein concentration using a BCA kit (P0010, Beyotime, China). Subsequently, proteins were separated through 10% SDS-PAGE (PG112, Epizyme, China) and transferred to a PVDF membrane, which was then blocked for 1 h with blocking liquid. The PVDF membranes underwent overnight incubation at 4°C with primary antibodies specifically targeting IQGAP3 (DF4389, Affinity, China), PLK1 (A2548, ABclonal, China), p-PI3K (AF3242, Affinity, China), PI3K (4249, CST, USA), p-AKT (4060, CST, USA), AKT (2920, CST, USA), and GAPDH (AC001, ABclonal, China) proteins. Subsequent to a single wash in 3x TBS, the membranes underwent an additional incubation step with HRP-conjugated goat anti-rabbit IgG secondary antibodies (ab6721, Abcam, GBR) for a duration of 1 h, conducted at room temperature. Detection of the blot’s signal was achieved employing the ChemiDoc chemiluminescence system (BIO-RAD, USA).
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4

Immunoblotting Procedure for Protein Detection

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For immunoblotting, parasites or host cells were lysed in lysis buffer (25 mM Tris–HCl, 200 mM NaCl, 10 mM NaF, 1 mM Na3VO4, 25 mM beta-glycerol phosphatase, 1% NP-40) supplemented with a protease inhibitor cocktail (Bimake, Shanghai, China) for 30 min on ice. Total cellular lysates were separated via SDS‒PAGE, and proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore), which were subsequently probed with the appropriate primary antibodies and corresponding secondary antibodies conjugated to horseradish peroxidase (HRP). The PVDF membranes were then subjected to treatment with enhanced chemiluminescence (ECL) reagents (Fdbio Science, Hangzhou, China), and the signals were immediately detected with a ChemiDoc™ chemiluminescence system (Bio-Rad, Hercules, USA).
The primary antibodies used were mouse anti-FLAG (1:1000), rabbit anti-HA (1:1000) and mouse anti-β-tubulin (1:2000) (Cell Signaling Technology). The following HRP-conjugated secondary antibodies were used: goat anti-mouse (1:5000) (Fdbio Science) and goat anti-rabbit (1:5000) (Fdbio Science).
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5

Expression and Purification of AMA1 Proteins

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pET30a-AMA1C, pET30a-AMA1N, and pET30a-AMA1 were separately transformed into the competent Escherichia coli strain Rosetta (DE3) pLysS (Novagen, Germany). They were then cultured overnight on lysogeny broth (LB) agar plates. A single colony was subcultured in LB medium, and protein expression was stimulated for 18 h at 23 °C by adding 1.0 mM isopropyl-d-1-thiogalactopyranoside (Aladdin, China). AMA1, AMA1N, and AMA1C were purified as previously described [20 (link)]. The three proteins were complexed for 24–36 h at 4 °C with renaturation buffers (pH 8.0–9.0; 0.1 M Tris–HCl, 0.5 mM L-Arg, 1 mM oxidized glutathione, and 1 mM reduced glutathione). Expression and purity of AMA1, AMA1N, and AMA1C were analyzed using sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE). The immunogenicity of proteins was subsequently assessed via western blotting by incubating the proteins with mouse His-tag monoclonal antibody (Proteintech Group, USA) (1:5000). Membranes were developed using BeyoECL Star (Beyotime Biotechnology, China) and visualized for acquisition using the ChemiDoc™ chemiluminescence system (Bio-Rad, Hercules, CA, USA). The concentrations of the purified proteins were quantified using a Bradford Protein Assay Kit (P0006, Beyotime Biotechnology, China).
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6

Western Blot Protein Detection

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Cells or tachyzoites were lysed in a radio-immunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology, China) supplemented with a protease inhibitor cocktail (Bimake, USA) at 4°C for 1 h. The whole cellular lysate was collected from the supernatants via centrifugation at 12,000 × g at 4°C for 10 min. The soluble proteins were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The membrane was probed with the primary antibody (1:1,000), incubated with the HRP-conjugated secondary antibody (1:5,000), and exposed to electrochemiluminescence (ECL) substrates (Fude, China). Signals were captured with the exposure meter ChemiDoc chemiluminescence system (Bio-Rad, USA).
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7

Western Blot Analysis of T. gondii Proteins

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Approximately 10 μg T. gondii soluble protein or 1 μg recombinant TgROP14 protein was loaded into each lane by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 12% gel and transferred to 0.22 μm PVDF membranes (Millipore, Darmstadt, Germany). Membranes were blocked with 5% (w/v) skim milk for 1 h at room temperature and probed with mouse anti-TgROP14 antibodies (1:2000) (prepared in this study), mouse anti-His antibody (1:1000) (Beyotime, Shanghai, China) or mouse anti-T. gondii antibodies (1:1000) (conserved in our laboratory), followed by horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:5000) (Fudebio, Hangzhou, China) for 1 h. Blots were exposed using ECL substrates (Fudebio, Hangzhou, China) and visualized using the ChemiDoc™ chemiluminescence system (Bio-Rad, Hercules, CA, USA).
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8

Western Blot Protein Extraction and Analysis

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Cells were harvested and suspended in ice-cold radioimmunoprecipitation assay buffer (RIPA) lysis buffer supplemented with a protease inhibitor cocktail, and incubated for 30 min on a horizontal shaker at 50 rpm under 4°C (Zhuang et al., 2020 (link)). Some protein samples were extracted with TRIzol reagent, resuspended in 1% SDS solution, and boiled in SDS loading buffer afterwards as described previously (Simões et al., 2013 (link); Wen et al., 2020 (link)). The soluble protein samples after 12,000 g × 10 min centrifugation were boiled for 10 min in SDS loading buffer, separated by SDS-PAGE, and transferred to 0.22 μm PVDF membrane (Millipore, USA). Membranes were blocked in 5% no-fat milk in Tris-buffered saline (TBS) supplemented with 0.5% Tween 20 for 2 h at 37°C. The incubation of primary antibodies in primary antibody dilution buffer took overnight at 4°C. The primary antibodies (1:5000) were detected using goat anti-mouse HRP-conjugated Ig G (H + L) (1:5000) or goat anti-rabbit HRP-conjugated Ig G (H + L) (1,5,000). Signals were documented by the ChemiDoc™ chemiluminescence system (BioRad, USA) after membranes were subjected to ECL substrates (FdBio Science, China) after fully rinsing. No image was changed in brightness and/or contrast. Densitometric analysis was measured by Image J (1.52v, National Institutes of Health, USA).
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9

Toxoplasma gondii Antigen Extraction

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Freshly harvested T. gondii tachyzoites, were washed thrice in phosphate buffered saline (PBS), sonicated 5 × 20 s at 5 kHz, and centrifuged at 12,000 g for 10 min to collect the supernatant containing T. gondii soluble antigens. About 10 µg soluble protein was loaded into each of lanes and transferred to 0.22 µm PVDF membranes (Millipore, Germany). Membranes were blocked with 5% (w/v) skim milk for 1 hour at room temperature and probed with appropriate primary antibodies, followed by horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Fudebio, China) for 1 hour. Blots were exposed using ECL substrates (Fudebio, China) and visualized using the ChemiDoc™ chemiluminescence system (Bio-Rad, USA).
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10

Western Blot Analysis of IQGAP3 and PIK3C2B

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In brief, RIPA lysis buffer (Beyotime Biotech, China) was used to extract total proteins from cells. Thirty micrograms of total protein from each sample was separated by 10% SDS-PAGE and then transferred onto a 0.45-μm PVDF membrane. The PVDF membranes were blocked with 5% BSA in TBST at room temperature for 2 h and then with primary antibodies against IQGAP3 (ab219354, at 1:2000 dilution, Abcam, USA), PIK3C2B (ab231122, at 1:1000 dilution, Abcam, USA), and GAPDH (M1310-2, at 1:3000 dilution; HuaBio Inc, China) overnight at 4°C. The membranes were incubated with the corresponding horseradish peroxidase (HRP)-labelled goat anti-rabbit or anti-mouse IgG antibody for 1h. After washing, the ChemiDoc chemiluminescence system (BIO-RAD, USA) was used to detect the signal of each blot.
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