35S::MYC and 35S::MYC-MdABI5 transgenic calli were subjected to ChIP experiments. Apple calli were crosslinked with formaldehyde and sonicated to disrupt chromatin. An anti-MYC antibody (Beyotime) was used for Chromatin immunoprecipitation-PCR (ChIP-PCR) as described by Hu et al.33 . qRT-PCR was performed to detect the enrichment of target DNA fragments. The primers used for ChIP-PCR are listed in Supplementary Table S1 .
Anti myc antibody
Manufactured by Beyotime
Sourced in China
The Anti-Myc antibody is a laboratory reagent used to detect and identify the presence of the Myc protein in various samples. It is a highly specific and sensitive tool for researchers working with Myc-tagged proteins or investigating Myc-related cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Emsa probe biotin labeling kit, by Beyotime (2 mentions)
Glutathione sepharose bead, by Thermo Fisher Scientific (2 mentions)
Lightshift chemiluminescent emsa kit, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pgadt7 vector, by Takara Bio (1 mentions)
Phis2 vector, by Takara Bio (1 mentions)
Anti β actin antibody, by Bioss Antibodies (1 mentions)
Ripa lysis buffer, by CoWin Biotech (1 mentions)
Anti flag antibody, by Beyotime (1 mentions)
Anti dmt1 antibody, by OriGene (1 mentions)
Anti ha antibody, by Beyotime (1 mentions)
Biospectrum imaging system, by Analytik Jena (1 mentions)
Protease inhibitor, by Roche (1 mentions)
4 protocols using anti myc antibody
1
MYC Protein-DNA Interactions in Apple Calli
2
ChIP-qPCR and EMSA Analysis of MdbHLH3 Transcription Factor
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The calli expressing 35S::Myc and 35S::MdbHLH3-Myc were subjected to ChIP-qPCR analysis. We used an anti-Myc antibody (Beyotime)6 (link). The immunoprecipitated DNA samples were used as templates for qPCR assays.
EMSA was conducted as described by Hu et al.6 (link). MdbHLH3-His recombinant protein was expressed in Escherichia coli strain BL21 and purified using glutathione sepharose beads (Thermo Scientific, San Jose, CA, USA). The EMSA probe biotin labeling kit (Beyotime) was used to label an oligonucleotide sequence corresponding to the MdDEP1 promoter, which was subsequently used for the binding assays with a LightShift chemiluminescent EMSA kit (Thermo).
EMSA was conducted as described by Hu et al.6 (link). MdbHLH3-His recombinant protein was expressed in Escherichia coli strain BL21 and purified using glutathione sepharose beads (Thermo Scientific, San Jose, CA, USA). The EMSA probe biotin labeling kit (Beyotime) was used to label an oligonucleotide sequence corresponding to the MdDEP1 promoter, which was subsequently used for the binding assays with a LightShift chemiluminescent EMSA kit (Thermo).
3
ChIP-qPCR and Yeast One-Hybrid Assay for MdbHLH3 Transcription Factor
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The ChIP (chromatin immunoprecipitation) experiment was performed as described by Hu et al. (2019 ). 35S::Myc and 35S::MdbHLH3‐Myc transgenic apple calli were used for the ChIP‐qPCR analysis, and an anti‐Myc antibody (Beyotime) was used for ChIP. All primers used for Chip‐PCR are listed in Table S1 . The full length of MdbHLH3 was ligated into the pGADT7 vector (Clontech). The MdcyMDH promoter 3 and promoter 4 region fragments were ligated into the pHIS2 vector (Clontech). 3‐AT (3‐amino‐1,2,4‐triazole) was used for screening. The yeast one‐hybrid assay was conducted as previously described (Li et al., ).
The EMSA was conducted as previously described (Hu et al.,2019 ). The CDSs of MdbHLH3 were cloned into the PET‐32a‐c vector. The MdbHLH3‐His recombinant protein was expressed in E. coli BL21 (DE3). The protein was purified using the Glutathione‐Sepharose beads (Thermo Scientific, San Jose, CA, USA). The EMSA Probe Biotin Labeling Kit (Beyotime) and the LightShift Chemiluminescent EMSA Kit (Thermo) were used for the subsequent EMSA. Briefly, the fusion protein MdbHLH3‐His and the oligonucleotide probe of the MdcyMDH promoter were incubated in a binding buffer for 20 min at room temperature. The unlabelled probes were used for probe competition (Hu et al., 2019 ).
The EMSA was conducted as previously described (Hu et al.,
4
Protein Extraction and Western Blot Analysis
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The collected cell samples were lysed in RIPA lysis buffer (ComWin Biotech Co, Beijing, China) prepared with protease inhibitors (Roche, Basel, Switzerland), and then were placed on ice for 30 min. The lysed cells were centrifuged at 12,000×g for 20 min at 4 °C, and the supernatant were collected. Protein concentrations were determined by the BCA Protein Assay Kit (Thermo Fisher Scientific, CA, USA). The supernatant (20 μg/well) was mixed with 5 × SDS-PAGE loading buffer and boiled in a 100 °C water bath for 5–10 min. The protein samples were subjected to gel electrophoresis and then transferred to PVDF membranes. After blocking with 10% skimmed milk powder for 2 h at room temperature, the membranes were incubated with anti-parkin antibody (1:1000, Millipore, Massachusetts, USA), anti-DMT1 antibody (1:800, OriGene, Rockville, MD, USA), anti-β-actin antibody (1:10,000, Bioss, Woburn, MA, USA), anti-HA antibody (1:2000, Beyotime Biotechnology, Shanghai, China), anti-Myc antibody (1:2000, Beyotime Biotechnology, Shanghai, China) and anti-Flag antibody (1:2000, Beyotime Biotechnology, Shanghai, China) overnight at 4 °C. Membranes were incubated with a corresponding secondary antibody (1:10,000, Bioss, Beijing, China) for 1 h at room temperature. Finally, blots were imaged using a BioSpectrum Imaging System (UVP, Upland, CA, USA) and quantified using ImageJ software.
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