Nuclear extracts were prepared, and electrophoretic mobility shift assay (EMSA) was performed as described previously [56 (link)–58 (link)] with some modifications. Briefly, IRDye infrared dye end-labeled oligonucleotides containing the consensus binding sequence for NFkB (5’- AGT TGA GGG GAC TTT CCC AGG C-3”) were purchased from Licor Biosciences. Six micrograms of nuclear extract was incubated with binding buffer and with infrared-labeled probe for 20 min. In the competition experiments, unlabeled oligonucleotide probe AP1 or NFkB was incubated with nuclear extract and binding buffer for 20 min. Subsequently, samples were separated on a 6% polyacrylamide gel in 0.25x TBE buffer (Tris borate-EDTA) and analyzed by the Odyssey Infrared Imaging System (LI-COR Biosciences).
Irdye infrared dye end labeled oligonucleotides
Manufactured by LI COR
IRDye infrared dye end-labeled oligonucleotides are synthetic DNA or RNA molecules with infrared dyes attached to their ends. These dyes enable the detection and visualization of the oligonucleotides in various laboratory applications.
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Lab products found in correlation
7 protocols using irdye infrared dye end labeled oligonucleotides
1
Electrophoretic Mobility Shift Assay for NF-κB
2
EMSA Analysis of CREB Activation
3
Nuclear Extract EMSA Assay
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Nuclear extracts were prepared, and EMSA was performed as described previously [7 , 26 (link), 27 (link)] with some modifications. Briefly, IRDye infrared dye end-labeled oligonucleotides containing the consensus binding sequence for CREB were purchased from Licor Biosciences. Six micrograms of nuclear extract was incubated with binding buffer and with infrared-labeled probe for 20 min. Subsequently, samples were separated on a 6% polyacrylamide gel in 0.25× TBE buffer (Tris borate-EDTA) and analyzed by the Odyssey Infrared Imaging System (LI-COR Biosciences).
4
Electrophoretic Mobility Shift Assay for NF-κB
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Nuclear extracts were prepared, and EMSA was performed as described previously (Pahan et al. 2001 (link); Rangasamy et al. 2018b (link)) with minor modifications. Briefly, IRDye infrared dye end-labeled oligonucleotides containing the consensus binding sequence for NF-κB were purchased from Licor Biosciences. Six micrograms of nuclear extract was incubated with binding buffer and with infrared-labeled probe for 20 min. Subsequently, samples were separated on a 6% polyacrylamide gel in 0.25 × TBE buffer (Tris borate-EDTA) and analyzed by the Odyssey Infrared Imaging System (LI-COR Biosciences).
5
EMSA Analysis of CREB Activation
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MN9D neuronal cells were treated with RNS60 and NS under serum-free condition. At different time periods of treatment, nuclear extracts were prepared to perform EMSA as described previously [54 (link),55 (link)] with some modifications. Briefly, IRDye infrared dye end-labeled oligonucleotides containing the consensus CREB DNA-binding sequence were purchased from Licor Biosciences and nuclear extract was incubated with infrared-labeled probe in binding buffer. Samples were separated on a 6% non-denaturing polyacrylamide gel in 0.25× TBE buffer (Tris borate-EDTA) and analyzed by the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA).
6
NF-κB Binding Assay with EMSA
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Nuclear extracts were prepared, and EMSA was performed as described previously (Pahan et al. 2001 (link); Jana et al. 2013 (link); Rangasamy et al. 2018 (link)) with minor modifications. We purchased IRDye infrared dye end-labeled oligonucleotides containing the consensus binding sequence for NF-κB from Licor Biosciences. Nuclear extract (6 µg) was incubated with binding buffer and with IRDye-labeled NF-κB probe for 20 min followed by separation on a 6 % polyacrylamide gel in 0.25× TBE buffer (Tris borate-EDTA) and analysis by the Odyssey Infrared Imaging System (LI-COR Biosciences).
7
Quantitative NF-κB DNA Binding Assay
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Electrophoretic mobility shift assay (EMSA) was performed using nuclear extracts45 (link)53 (link)54 (link) with some modifications. IRDye infrared dye end-labeled oligonucleotides (5′-AGT TGA GGG GAC TTT CCC AGG C-3′) for NF-κB was procured from Licor Biosciences. Six micrograms of nuclear extract was incubated with infrared-labeled probe in binding buffer for 20 min. Further, samples were separated on a 6% polyacrylamide gel electrophoresis with 0.25 × TBE buffer (Tris borate-EDTA). Images were captured and analyzed with Odyssey Infrared Imaging System (LI-COR Biosciences).
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