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Lightshift chemiluminescent electrophoretic mobility shift assay kit

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The LightShift Chemiluminescent EMSA Kit is a tool used for the detection and analysis of protein-DNA interactions. It employs a chemiluminescent detection method to visualize the binding of transcription factors or other DNA-binding proteins to their target DNA sequences.

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Lab products found in correlation

Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (4 mentions) Chemiluminescent nucleic acid detection module, by Thermo Fisher Scientific (2 mentions) Ne per nuclear extraction reagent, by Thermo Fisher Scientific (2 mentions) Glutathione sepharose 4b column, by GE Healthcare (1 mentions) Hoechst 33258, by Thermo Fisher Scientific (1 mentions) Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions) Oridonin, by Merck Group (1 mentions) 3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide, by Merck Group (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Alexa fluor 488 donkey anti mouse igg h l antibody, by Thermo Fisher Scientific (1 mentions) Fitc annexin 5 apoptosis detection kit, by BD (1 mentions) Fluor 594 donkey anti rabbit igg h l antibody, by Thermo Fisher Scientific (1 mentions) Biodyne b nylon membrane, by Thermo Fisher Scientific (1 mentions) T100 thermal cycler, by Bio-Rad (1 mentions) Retardation gel, by Thermo Fisher Scientific (1 mentions) Anti nf κb p65, by Santa Cruz Biotechnology (1 mentions) Biotin labeled probe, by Sangon (1 mentions) Anti relb, by Santa Cruz Biotechnology (1 mentions) Anti stat3, by Santa Cruz Biotechnology (1 mentions) Glutathione agarose beads, by BD (1 mentions)

Spelling variants of this product

Electrophoretic Mobility Shift Assay kit (11 citations) LightShift Chemiluminescent kit (6 citations)

19 protocols using lightshift chemiluminescent electrophoretic mobility shift assay kit

1

Purification and Characterization of SiARDP-GST and SiAREB1-GST Proteins

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SiARDP-GST and SiAREB1-GST vectors were constructed and transferred into E. coli (BL21) by Li et al. (2014) (link). After induction, the fusion proteins were purified trough Glutathione Sepharose 4B column (GE, USA). Oligonucleotides (P1-R, P2-R, and P3-R) and their reverse complementary oligonucleotides (P1-F, P2-F, and P3-F, respectively), which were labeled with biotin, were synthesized. Meanwhile, the oligonucleotides (competitor2-1R, competitor2-1F, competitor2-2R, and competitor2-2F) for competitors were synthesized. All of these sequences are shown in Supplementary Table S1. Double-stranded DNA was obtained by heating oligonucleotides at 92°C for 30 s and annealing at 30°C overnight. The gel-shift assay was performed following the manufacturer’s protocol for the LightShift Chemiluminescent Electrophoretic Mobility Shift Assay (EMSA) Kit (Thermo, USA).
Pan Y., Li J., Jiao L., Li C., Zhu D, & Yu J. (2016). A Non-specific Setaria italica Lipid Transfer Protein Gene Plays a Critical Role under Abiotic Stress. Frontiers in Plant Science, 7, 1752.
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2

PPRE Consensus Oligonucleotide EMSA Protocol

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The Light Shift Chemiluminescent Electrophoretic Mobility Shift Assay (EMSA) kit (Thermo Fisher Scientific, 20148) was used to detect DNA-protein interactions. The sequences of peroxisome proliferator-activated receptor response element (PPRE) consensus oligonucleotides were as follows: forward, 5′-CAAAACTAGGTCAAAGGTCA-3′; reverse, 5′-GTTTTGATCCAGTTTCCAGT-3′.
Oligonucleotides were labeled with biotin at the 5′-end. The reaction mixture was subjected to PAGE and transferred to nylon membranes after incubation with an oligonucleotide probe for 20 min, which was immediately cross-linked using a ultraviolet transilluminator. Chemiluminescence was then used to detect the bands.
Zhong Y., Wang Y., Li X., Qin H., Yan S., Rao C., Fan D., Liu D., Deng F., Miao Y., Yang L, & Huang K. (2023). PRMT4 Facilitates White Adipose Tissue Browning and Thermogenesis by Methylating PPARγ. Diabetes, 72(8), 1095-1111.
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3

Oridonin Cytotoxicity in 4T1 Breast Cancer

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Oridonin (purity ≥ 98%) was purchased from Sigma-Aldrich China Inc. (Shanghai, China). The human breast cancer cell line 4T1 were obtained from the American Type Culture Collection (Rockville, Maryland, USA). Cell counting kit-8 (CCK-8) was provided by Dojindo Laboratories (Kumamoto, Japan). Hoechst 33258, the ApoDETECT annexin V–fluorescein isothiocyanate (FITC) apoptosis detection kit, the Light Shift chemiluminescent electrophoretic mobility shift assay (EMSA) kit, and NE-PER nuclear and cytoplasmic extraction reagents were obtained from ThermoFisher Scientific Inc. (Waltham, Massachusetts, USA). The cells were cultured in DMEM medium (Hyclone, Beijing, China) enriched with 10% fetal bovine serum (Hyclone) at 37 °C and 5% CO2.
Xia S., Zhang X., Li C, & Guan H. (2017). Oridonin inhibits breast cancer growth and metastasis through blocking the Notch signaling. Saudi Pharmaceutical Journal : SPJ, 25(4), 638-643.
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4

Nuclear Protein-DNA Binding Assay

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Nuclear proteins were extracted from cells using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific). Then, nuclear extracts were incubated with synthetic 3′ biotin-labeled 23-bp oligonucleotides by using a LightShift Chemiluminescent Electrophoretic Mobility Shift Assay (EMSA) Kit (Thermo Fisher Scientific). The oligonucleotide sequences are shown in Supplementary Table 4 (available online). The binding reactions were subjected to electrophoresis on a 6% polyacrylamide gel, and the products were detected by a chemiluminescent reaction with a stabilized streptavidin-horseradish peroxidase conjugate. Unlabelled probes were added to the reaction at a 200-fold excess for competition assays.
Fan L., Wang H., Ben S., Cheng Y., Chen S., Ding Z., Zhao L., Li S., Wang M, & Cheng G. (2024). Genetic variant in a BaP-activated super-enhancer increases prostate cancer risk by promoting AhR-mediated FAM227A expression. Journal of Biomedical Research, 38(2), 149-162.
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5

Characterizing Transcription Factor Binding

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NE-PER Nuclear and Cytoplasmic Extraction Reagents, LightShift Chemiluminescent Electrophoretic mobility shift assay (EMSA) Kit, and Chemiluminescent Nucleic Acid Detection Module were purchased from Thermo Scientific. Purified biotin-labeled probes were purchased from Sangon Biotech. Cells were treated with DMSO or TPA, and then nuclear extracts were isolated. For binding assays, 3 μg nuclear extracts were incubated with 20 fmol probes at room temperature for 20 min in 20 μl reaction buffers. Electrophoreses, transfer to a nylon membrane, and UV cross-linking were performed according to the manufacturer’s protocol. For confirming the specificity of the transcription factor binding to the probe, the antibodies against c-Jun or Sp1 (Santa Cruz) and 3 μg nuclear extracts were incubated for 20 min at room temperature before adding the purified biotin-labeled probe for another 20 min at room temperature. Oligonucleotide probes used in EMSA were named as wild-type (WT), Am, Sm, or Sam, and their sequences corresponded to the human VIL2 V1 promoter region from -87 to -46 bp (Table 2).
Zhang X.D., Xie J.J., Liao L.D., Long L., Xie Y.M., Li E.M, & Xu L.Y. (2015). 12-O-Tetradecanoylphorbol-13-Acetate Induces Up-Regulated Transcription of Variant 1 but Not Variant 2 of VIL2 in Esophageal Squamous Cell Carcinoma Cells via ERK1/2/AP-1/Sp1 Signaling. PLoS ONE, 10(4), e0124680.
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6

Nuclear Protein Isolation and NF-κB Analysis

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Nuclear proteins were isolated from ischaemic cerebral hemispheres as described previously 9 (link),10 (link) and NF-κB binding activity was examined by Light Shift Chemiluminescent electrophoretic mobility shift assay (EMSA) kit (Thermo Scientific, Waltham, MA, USA) according to the instructions of the manufacturer.
Zhang X., Ha T., Lu C., Lam F., Liu L., Schweitzer J., Kalbfleisch J., Kao R.L., Williams D.L, & Li C. (2014). Poly (I:C) therapy decreases cerebral ischaemia/reperfusion injury viaTLR3-mediated prevention of Fas/FADD interaction. Journal of Cellular and Molecular Medicine, 19(3), 555-565.
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7

Daidzin-Induced Apoptosis Assay

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Daidzin (DDZ) was purchased from Weikeqi Biological Technology (Chengdu, Sichuan. China). Bovine serum albumin (BSA) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO). Alexa Fluor® 488 donkey anti-mouse IgG (H+L) antibody and Fluor® 594 donkey anti-rabbit IgG (H+L) antibody were obtained from Life Technologies (Grand Island, NY, USA). A LightShift® Chemiluminescent electrophoretic mobility shift assay (EMSA) kit was purchased from Thermo Fisher Scientific Inc. An FITC Annexin V Apoptosis Detection Kit was purchased from BD Pharmingen™ (BD Biosciences, Becton Dickinson, Franklin Lakes, NJ, USA).
Yang M.H., Jung S.H., Chinnathambi A., Alahmadi T.A., Alharbi S.A., Sethi G, & Ahn K.S. (2019). Attenuation of STAT3 Signaling Cascade by Daidzin Can Enhance the Apoptotic Potential of Bortezomib against Multiple Myeloma. Biomolecules, 10(1), 23.
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8

Oligonucleotide Binding Assay for SNP

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Nuclear protein was extracted from HEK293 and THP-1 cell lines in different experiments. Thirty mer oligonucleotides flanking rs2069837 with A/G allele (5′-TGCCAGGCACTTTAA/GATAAATATTGTGTCT-3′) and their complementary oligonucleotides (5′-AGACACAATATTTATT/CTAAAGTGCCTGGCA-3′) were synthesised with or without 5′ end biotin label (Integrated DNA Technologies). The complementary oligomers were annealed into corresponding double-stranded DNA on Bio-Rad T100 Thermal Cycler (95°C for 5 min; step cooling (95°C (−1°C/cycle), 70 cycles; holding at 4°C). The biotin-labelled probes with A or G were incubated with nuclear extract in a binding buffer (50 ng/μl Poly (dI•dC), 2.5% glycerol, 0.05% nonidet P-40, 5 mM MgCl2, 10 mM EDTA, 20 μL system) using LightShift Chemiluminescent electrophoretic mobility shift assay (EMSA) Kit (Thermo Fisher Scientific) for 20 min at room temperature before separating on a 6% retardation gel (Thermo Fisher Scientific). For competitive EMSA, different fold excess (1–400×) of unlabeled DNA were added. After electrophoresis, the protein and DNA complexes were transferred to biodyne B nylon membrane (Thermo Fisher Scientific) and observed by chemiluminescent detection methods postultraviolet cross-linking.
Kong X, & Sawalha A.H. (2019). Takayasu arteritis risk locus in IL6 represses the anti-inflammatory gene GPNMB through chromatin looping and recruiting MEF2–HDAC complex. Annals of the rheumatic diseases, 78(10), 1388-1397.
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9

Investigating Transcription Factor Binding

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LightShift Chemiluminescent Electrophoretic mobility shift assay (EMSA) kit and Chemiluminescent Nucleic Acid Detection Module were purchased from Thermo Fisher Scientific. Purified biotin-labeled probes were synthesized from Sangon Biotech, Shanghai, China. The probe sequences were in the Supplementary Table S3. Nuclear extracts of KYSE150 cells were isolated by NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific). For binding assays, 2 μg nuclear extracts were incubated with 20 fmol probes at room temperature for 25 min in 20 μL reaction buffers. Electrophoreses, transferring to a nylon membrane and crosslinking at high temperature (120 °C), were performed in accordance with the manufacturer’s protocol. To confirm the specificity of the binding between transcription factors and the probes, antibodies anti-STAT3, anti-NF-κB p65, anti-Rel-B (Santa Cruz), and 2 μg nuclear extracts were incubated for 10 min at 4 °C.
Long L., Pang X.X., Lei F., Zhang J.S., Wang W., Liao L.D., Xu X.E., He J.Z., Wu J.Y., Wu Z.Y., Wang L.D., Lin D.C., Li E.M, & Xu L.Y. (2018). SLC52A3 expression is activated by NF-κB p65/Rel-B and serves as a prognostic biomarker in esophageal cancer. Cellular and Molecular Life Sciences, 75(14), 2643-2661.
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10

NF-κB and AP-1 Binding Assay

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RAW264.7 cells were pre-treated with 2.0 μg/ml rCC16 for 2 h, followed by LPS treatment for another 1 h. Then, 5.0 μg of nuclear extract was incubated with 20 nM biotin-labeled double-stranded oligonucleotide probes (Beyotime, China) containing the consensus sequence for the NF-κB-DNA or AP-1-DNA-binding site for 20 min at room temperature and then separated on a non-denaturing 6% (w/v) polyacrylamide gel. The biotin-labeled oligonucleotide probes were transferred from the polyacrylamide gel onto a nylon membrane and then detected using the LightShift Chemiluminescent electrophoretic mobility shift assay (EMSA) kit (Thermo Scientific) according to the manufacturer's instructions.
Pang M., Yuan Y., Wang D., Li T., Wang D., Shi X., Guo M., Wang C., Zhang X., Zheng G., Yu B, & Wang H. (2017). Recombinant CC16 protein inhibits the production of pro-inflammatory cytokines via NF-κB and p38 MAPK pathways in LPS-activated RAW264.7 macrophages. Acta Biochimica et Biophysica Sinica, 49(5), 435-443.
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