Biotin 3 end dna labeling kit

Cells from a 10-cm dish were resuspended in PBS. The cytoplasmic and nuclear fraction were collected using nuclear and cytoplasmic protein extraction kit (P0028, Beyotime, Hangzhou, China). For analysis of ZEB1 DNA binding, EMSA was used and performed as previously described [49 ]. Double-stranded oligonucleotide probes were synthesized (Beyotime, Hangzhou, China) to contain the E-box site sequences from the promoters of the indicated target genes (ATM). The oligos were end labeled with biotin using biotin 3′end DNA labeling kit (GS008, Beyotime, Hangzhou, China). EMSA was performed by incubating 5 μg of nuclear extract with 4 nmol biotin-labeled DNA probe. Absence of nuclear extract, competition with 100-fold molar excess unlabeled DNA prob or non-specific oligo served as controls. Supershift studies were performed by pre-incubation with the specified antibodies for 30 min. All EMSA assays were performed by chemiluminescent EMSA kit (GS009, Beyotime, Hangzhou, China). Complexes were separated by electrophoresis on non-denaturing 6.5% acrylamide gel. Sequences of EMSA probes are provided as follow: E-box: forward 5′-TGGCATTTCACACCTCTACACTGGACG′, reverse 5′-CGTCCAGTGTAGAGGTGTGAAATGCCA-3′; Non-specific: forward 5′-TCGAGTTGATGTAACCGACTCAGGCACT′, reverse 5′-AGTGCCTGAGTCGGTTACATCAACTCGA-3′.

Electrophoresis mobility shift assay was performed as described previously (Ma et al., 2009). Briefly, the CDS of OsPIL15 was cloned into the expression vector pET28a and the constructs were transformed into E. coli BL21 (DE3). For purification of the fusion protein, the cells were lysed using an ultrasonic cell disruptor, and the His‐tagged fusion proteins were purified with Ni‐NTA resin (Qiagen, Valencia, CA) according to the manufacturer's instructions.
Oligonucleotides (Table S5) were synthesized and labeled using the Biotin 3′ End DNA Labeling Kit (Beyotime, Shanghai, China). DNA probes were obtained by annealing two complementary oligonucleotides. The DNA gel mobility shift assay was performed using the EMSA kit (Beyotime, Shanghai, China) following the manufacturer's protocol. Briefly, the probes were incubated for 30 min with OsPIL15 protein in binding buffer at room temperature then the DNA–protein complexes were separated on native polyacrylamide gels (6%). The results were quantified using ImageJ software.

The crp gene was inserted into the downstream of a sequence encoding hexa-histidine in pET28a so that Escherichia coli host produces His₆-CRP, and then, the recombinant protein was purified using a Ni–NTA agarose column (Spriestersbach et al., 2015 (link)). The putative promoter region fragments of tolB, ftsI, or rcsF were amplified and labeled using the biotin 3′ end DNA labeling kit (Beyotime, China). The labeled single-stranded probes were annealed with an annealing reagent (Beyotime) and incubated with increasing amounts of His₆-CRP protein (0, 0.6, 1, and 2 μM). After incubation at room temperature for 30 min, the mixtures were analyzed using 6% native polyacrylamide gel containing 1 nmol cAMP. The biotin-labeled DNA was detected by the Chemiluminescence EMSA Kit (Thermo Fisher Scientific, United States).