Nuclear proteins were extracted using NE-PERⓇ Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, Rockford, IL, USA). Extraction was performed according to the manufacturer’s protocol. Oligonucleotides containing the κ-chain (κB, 5’-CCGGTTAACAGAGGGGGCTTTCCGAG-3’) binding site were synthesized and used as probes for the gel retardation assays. The NF-κB oligonucleotide was labeled with [α-32P]dCTP using a Rediprime II DNA Labeling System (Amersham Life Science, Amersham, UK). In competition assays, a 100-fold excess of cold κB oligonucleotide was added. The DNA-protein complexes were analyzed via electrophoresis on a 4% polyacrylamide gel. After electrophoresis, the gel was dried and examined using autoradiography.
Rediprime 2 dna labeling system
Manufactured by Cytiva
Sourced in United Kingdom, United States
The Rediprime II DNA Labeling System is a ready-to-use kit for the random prime labeling of DNA fragments. It allows the incorporation of radioactive or non-radioactive labels into DNA probes for use in various hybridization techniques.
Automatically generated - may contain errors
2 protocols using rediprime 2 dna labeling system
1
Nuclear Protein Extraction and EMSA
2
Quantitative Northern Blot Analysis of HCV RNA
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Total RNA (5 μg), which was extracted from Huh7 cells and JFH1 RNA-transfected Huh7 cells using Trizol reagent (Invitrogen), was resolved on a denaturing agarose (0.7%) gel and transferred onto a positive charged nylon membrane (Roche Diagnostics, Mannheim, Germany) and fixed to the membrane by UV-crosslinking. HCV (JFH1) and GAPDH probes were generated by random labeling of HCV and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA fragments prepared by RT-PCR using the following primer sets: sense primer JFH1 F (5′-CCAACCTGCTCATGGAGG-3′), antisense primer JFH1 R (5′-TCCACGCAGAACACCTCA-3′), sense primer GAPDH F (5′-GAAGGTGAAGGTCGGAGTC-3′), and antisense primer GAPDH R (5′-GGGACTCCCCAGCAGTG-3′). The PCR-amplified DNA fragments were radiolabeled using [α-32P] dCTP and Rediprime II DNA labeling system (Amersham Biosciences, PA, USA). Membranes were pre-hybridized for 30 min and then hybridized with HCV-specific radiolabeled probes overnight. After washing, membranes were quantified using PhosphorImager and normalized to GAPDH. Uncropped images of northern blots are provided in Supplementary Fig. 7b .
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