Protein-DNA Binding Assay with OsGIF1 and SLR1

EMSA was performed as previously described with minor modifications39 (link). Full-length OsGIF1 and SLR1 cDNAs were amplified and cloned into the pCold-TF vector (Takara). His-OsGIF1 and His-SLR1 recombinant proteins were purified using Ni-NTA agarose (QIAGEN, 30210), following the manufacturer’s instructions. GST (Glutathione S-transferase) and GST-OsGRF4 recombinant protein were expressed in the Escherichia coli BL21 (DE3) strain and then purified using Glutathione Sepharose 4B beads (GE Healthcare, 17-0756-01). 42 bp DNA probes were artificially amplified and labelled using a biotin label kit (Biosune). DNA gel shift assays were performed using the LightShift Chemiluminescent EMSA kit (Thermo Fisher Scientific, 20148). Relevant primer sequences are given in Supplementary Information Table 8.

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Li S., Tian Y., Wu K., Ye Y., Yu J., Zhang J., Liu Q., Hu M., Li H., Tong Y., Harberd N.P, & Fu X. (2018). Modulating plant growth-metabolism coordination for sustainable agriculture. Nature, 560(7720), 595-600.

Publication 2018

pCold-TF vector Ni-NTA agarose Glutathione Sepharose 4B beads LightShift Chemiluminescent EMSA kit

Agarose Biotin Cdnas Dna probes Emsa Escherichia coli Glutathione Glutathione s transferase Primer Recombinant protein Sepharose 4b Strain Vector

Corresponding Organization :

Other organizations : Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Hebei Academy of Agriculture and Forestry Sciences, University of Oxford, University of Chinese Academy of Sciences

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Variable analysis

independent variables

Cloning of full-length OsGIF1 and SLR1 cDNAs into pCold-TF vector
Expression and purification of His-OsGIF1 and His-SLR1 recombinant proteins
Expression and purification of GST and GST-OsGRF4 recombinant proteins

dependent variables

DNA binding activity of the recombinant proteins (measured using EMSA)

control variables

EMSA protocol based on previously described methods with minor modifications
Use of Ni-NTA agarose and Glutathione Sepharose 4B beads for protein purification
Use of 42 bp DNA probes amplified and labeled using a biotin label kit
Use of LightShift Chemiluminescent EMSA kit for DNA gel shift assays

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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