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Emsa probe biotin labeling kit

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The EMSA Probe Biotin Labeling Kit is a laboratory tool designed for the labeling of DNA or RNA probes with biotin. The kit provides the necessary reagents and protocols for efficient biotin labeling of nucleic acid probes, enabling their use in various downstream applications such as electrophoretic mobility shift assays (EMSAs).

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Lab products found in correlation

Chemiluminescent emsa kit, by Beyotime (29 mentions) Lightshift chemiluminescent emsa kit, by Thermo Fisher Scientific (9 mentions) Nylon membrane, by Beyotime (7 mentions) Emsa gel shift kit, by Beyotime (6 mentions) Glutathione sepharose bead, by Thermo Fisher Scientific (4 mentions) Emsa gel shift binding buffer, by Beyotime (3 mentions) Chemiluminescent biotin labeled nucleic acid detection kit, by Beyotime (3 mentions) Acetyl phosphate, by Merck Group (3 mentions) Positively charged nylon membrane, by Beyotime (3 mentions) Charged nylon membrane, by Beyotime (2 mentions) Streptovidin hrp, by Beyotime (2 mentions) Anti myc antibody, by Beyotime (2 mentions) Proteinlsor ni nta resin, by Transgene (2 mentions) Nuclear and cytoplasmic protein extraction kit, by Beyotime (2 mentions) Lightshift chemiluminescent emsa kit, by Beyotime (2 mentions) Emsa kit, by Beyotime (2 mentions) Gst tag protein purification kit, by Beyotime (2 mentions) Chemdoc touch imaging system, by Bio-Rad (2 mentions) His tagged protein purification kit, by Beyotime (1 mentions) Oligonucleotide probes, by Sangon (1 mentions)

Close spelling variants of this product

Biotin-labeling kit (4 citations) EMSA kit (64 citations)

68 protocols using emsa probe biotin labeling kit

1

Biotin-labeled Probe Preparation and EMSA

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The biotin‐labeled probes were prepared as described in the instructions for the EMSA Probe Biotin Labeling Kit (Beyotime). Generally, we firstly amplified the unlabeled fragments with specific primers (Table S3) and purified the PCR products using a kit (FastPureR Gel DNA Extraction Mini Kit; Vazyme). The purified PCR products were denatured at 95°C for 2 min to create single‐stranded DNA fragments. These fragments were incubated with biotin‐11‐dUTP at 37°C for 30 min to obtain biotin‐labeled single‐stranded probes. The labeled probes were then renatured to double‐stranded probes following the manufacturer's instructions (EMSA Probe Biotin Labeling Kit; Beyotime).
For the binding assay, the purified MBP‐ARF8 protein was incubated with 0.5 μM specific probes in binding buffer (10 mM Tris, 0.2 mM EDTA, 20 mM KCl, 1% glycerol, 0.02% TritonX‐100, LightShift Poly (dI‐dC; Invitrogen)). For the competition assay, 2–4 μM competitors or cold competitors were added to the reaction. The samples were incubated at 25°C for 20 min. Then the reaction mixtures were separated via a 6% native polyacrylamide gel and transferred to a nylon membrane (Amersham Hybond™‐N+; GE, Chengdu, China). Later steps followed the instructions of the Chemiluminescent EMSA Kit (Beyotime). Luminescence intensity was detected by using a low‐light cooled charge‐coupled device imaging apparatus.
Feng Q., Wang H., Yang X., Hu Z., Zhou X., Xiang L., Xiong X., He X., Zhu Y., Li G., Zhao J., Ji Y., Hu X., Pu M., Zhou S., Zhao Z., Zhang J., Huang Y., Fan J., Wang W, & Li Y. (2022). Osa‐miR160a confers broad‐spectrum resistance to fungal and bacterial pathogens in rice. The New Phytologist, 236(6), 2216-2232.
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2

EMSA Protocol for Detecting PPARα Binding

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EMSA was carried out as previously described [56 (link)]. In brief, the lysates of HEK293T cells, transfected with pcDNA3.1-Flag-PPARαb were prepared for DNA/protein conjugation reactions. The EMSA Probe Biotin Labeling Kit (Beyotime, Shanghai, China) was used to label mutated and wild-type oligonucleotides (Table S1) for EMSA biotin-labeled probes, according to the manufacturer′s instructions. DNA/protein binding reactions were performed using an EMSA/Gel-Shift Kit (Beyotime, China) at 25 °C, based on the manufacturer′s instructions. To detect the specificity of DNA/protein binding reactions, competition assays were carried out with 100 × excessive unlabeled wild-type or mutated probes. Completed reactions were separated on nondenaturing 4% PAGE gels for 20 min. The proteins were developed by autoradiography using a LightShift® Chemiluminescent EMSA Kit (Pierce, USA).
Zhu K.C., Song L., Guo H.Y., Guo L., Zhang N., Liu B.S., Jiang S.G, & Zhang D.C. (2018). Identification of Fatty Acid Desaturase 6 in Golden Pompano Trachinotus Ovatus (Linnaeus 1758) and Its Regulation by the PPARαb Transcription Factor. International Journal of Molecular Sciences, 20(1), 23.
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3

Protein-DNA Interaction Assay Using Custom Cell-Free Expression

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The flag-OsNF-YB1 protein was expressed using Gzl Custom Cell Free Protein Expression Kit (GZL bioscience). DNA probes in a length of 59 nt were commercially synthesized by GENEWIZ Biological Technology and labeled with an EMSA Probe Biotin Labeling Kit (Beyotime, Cat No. GS008). DNA binding was performed in a 10-L reaction volume containing EMSA/Gel-shift binding buffer (Beyotime), 2 nmol biotin-labeled probe, and 5 nmol purified recombinant protein. Non-labeled DNA oligos were used as competitor. Recombinant protein was pre-incubated with the EMSA/Gel-shift binding buffer for 20 min at 25°C prior to the addition of the biotin-labeled probe and further incubated at 25°C for 20 min. A 6% (W/V) polyacrylamide gel was pre-run for 30 min, and then the binding reaction is subjected to gel electrophoresis. The DNA probes were then transferred to a charged nylon membrane (Beyotime), detected by streptovidin-HRP (Beyotime), and finally visualized using enhanced chemiluminescence (Pierce).
Zhang D., Zhang M, & Liang J. (2021). RGB1 Regulates Grain Development and Starch Accumulation Through Its Effect on OsYUC11-Mediated Auxin Biosynthesis in Rice Endosperm Cells. Frontiers in Plant Science, 12, 585174.
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4

Purification and EMSA of PeSTZ1 Fusion Proteins

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The HIS‐PeSTZ1 fusion proteins were obtained from in vitro prokaryotic expression. The cDNAs encoding full‐length PeSTZ1 were cloned into PET28a to generate His‐fusion recombinant vectors, which were then expressed in Escherichia coli BL21. Then, 0.2 mm isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) was applied to induce the protein. His‐fusion proteins were purified with a His‐Tagged Protein Purification kit (Beyotime, GS008 and GS009, China) according to the manufacturer's instructions.
The PeAPX2 promoter fragment containing CATTAACACTG motifs was synthesized by Sangon (Beijing, China). The EMSA Probe Biotin Labeling kit and a Chemiluminescent EMSA kit (GS008 and GS009; Beyotime Biotechnology, Shanghai, China) were used for the subsequent EMSA assays, which were performed according to the manufacturer's protocols. Briefly, biotin‐labelled probes and fusion proteins were mixed in a binding buffer for 30 min at 22 °C. The unlabelled probes were used for probe competition, and the HIS protein was used as a negative control.
He F., Li H., Wang J., Su Y., Wang H., Feng C., Yang Y., Niu M., Liu C., Yin W, & Xia X. (2019). PeSTZ1, a C2H2‐type zinc finger transcription factor from Populus euphratica, enhances freezing tolerance through modulation of ROS scavenging by directly regulating PeAPX2. Plant Biotechnology Journal, 17(11), 2169-2183.
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5

Characterization of BnTGA7 DNA-Binding Activity

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The full-length CDS of BnTGA7 was cloned into the pGEX6P-1 vector to generate the glutathione S-transferase (GST)–BnTGA7 fusion protein, which was expressed in Escherichia coli strain BL21. Expression and purification of the GST–BnTGA7 protein were performed according to the manufacturer’s instructions (Transgen, Beijing, China). A 30 bp DNA sequence containing the TGACG sequence was synthesized by Beijing Qingke Biotechnology and labeled with an EMSA Probe Biotin Labeling Kit (Beyotime, Shanghai, China). EMSA was performed using a chemiluminescent EMSA kit according to the manufacturer’s instructions (Beyotime). In brief, GST–BnTGA7 and the labeled probe were incubated at 25 °C for 20 min in a reaction system containing EMSA/gel-shift binding buffer. For the cold competition, 200-fold unlabeled probe was added to the reaction mixtures. These reaction mixtures were loaded on an 8% native PAGE gel; after transferring to a nylon membrane, cross-linking, and blocking the proteins, the signals were detected with a chemiluminescent EMSA kit.
Lin L., Fan J., Li P., Liu D., Ren S., Lin K., Fang Y., Lin C., Wang Y, & Wu J. (2022). The Sclerotinia sclerotiorum-inducible promoter pBnGH17D7 in Brassica napus: isolation, characterization, and application in host-induced gene silencing. Journal of Experimental Botany, 73(19), 6663-6677.
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6

MdFLP Protein-DNA Interaction Analysis

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EMSA was conducted according to Xie et al. [40 (link)]. MdFLP was inserted into the pGEX4T-1 plasmid. The MdFLP-GST plasmid was expressed in BL21 and purified using glutathione Sepharose beads. Three oligonucleotide probes of the MdPIN3 and MdPIN10 promoters were labeled using an EMSA probe biotin labeling kit (Beyotime). The mutant probes were labeled and contained one mutated nucleotide. The EMSAs were conducted following the manufacturer’s instructions (Thermo Scientific). The binding specificity was also examined by competition with a fold excess of unlabeled oligonucleotides. The primers used for EMSA are listed in Additional file 2: Table S1.
Wang Z., Li J., Mao Y., Zhang M., Wang R., Hu Y., Mao Z, & Shen X. (2019). Transcriptional regulation of MdPIN3 and MdPIN10 by MdFLP during apple self-rooted stock adventitious root gravitropism. BMC Plant Biology, 19, 229.
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7

Purification and DNA Binding Assay of bZIP Proteins

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The GST-O3104–176aa protein containing the bZIP domain structure was purified. The EMSA probes of BIP1, PDIL1-1, GBSS1, and OsGluA2 were commercially synthesized and labeled using the EMSA Probe Biotin Labeling Kit (Beyotime, Shanghai, China), and non-labeled probes were used as competitors. Probe sequences are listed in Supplemental Table 1. The DNA binding reaction was performed for 20 min at 25°C, and the products were then electrophoresed using 6% acrylamide gels. The DNA probes were transferred to a nylon membrane. Finally, the oligo bands were detected using the LightShift Chemiluminescent EMSA Kit and streptavidin-horseradish peroxidase (Beyotime).
Cao R., Zhao S., Jiao G., Duan Y., Ma L., Dong N., Lu F., Zhu M., Shao G., Hu S., Sheng Z., Zhang J., Tang S., Wei X, & Hu P. (2022). OPAQUE3, encoding a transmembrane bZIP transcription factor, regulates endosperm storage protein and starch biosynthesis in rice. Plant Communications, 3(6), 100463.
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8

Characterizing E75 Binding to Chi2 Promoter

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EMSA was performed to check the interaction of E75 with putative E75 binding sites in the promoter region of Chi2. Oligonucleotide probes were synthesized (Sangon Biotech, Shanghai, China), and labeled with biotin using an EMSA Probe Biotin Labeling Kit (Beyotime, Shanghai, China; GS008). EMSA was performed using a Chemiluminescent EMSA Kit (Beyotime; GS009), as described by the manufacturer. Generally, 2 μg of purified recombinant E75 was incubated with 5 ng of the probes at 25°C for 20 min in a binding buffer. In the competition binding assays, excess unbiotinylated probes were added to the mixture. The samples were analyzed on 6% acrylamide gels in 0.5 × Tris-borate-EDTA buffer and then electroblotted onto a nylon membrane. The labeled DNA was detected and visualized using a chemiluminescent biotin-labeled nucleic acid detection kit.
Wang X.X., Ding M.J., Gao J., Zhao L., Cao R, & Wang X.W. (2024). Modulation of host lipid metabolism by virus infection leads to exoskeleton damage in shrimp. PLOS Pathogens, 20(5), e1012228.
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9

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).
Hu D.G., Sun C.H., Zhang Q.Y., Gu K.D, & Hao Y.J. (2020). The basic helix-loop-helix transcription factor MdbHLH3 modulates leaf senescence in apple via the regulation of dehydratase-enolase-phosphatase complex 1. Horticulture Research, 7, 50.
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10

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).
Yu J., Gu K., Sun C., Zhang Q., Wang J., Ma F., You C., Hu D, & Hao Y. (2020). The apple bHLH transcription factor MdbHLH3 functions in determining the fruit carbohydrates and malate. Plant Biotechnology Journal, 19(2), 285-299.
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