Protease Sensitivity Assay of AtHYL1

The protease sensitivity assay using bacterially purified AtHYL1FL and AtHYL1N terminal was performed according to previous reports [35 (link),36 (link)]. The samples were loaded on 12–15% SDS-PAGE, and the protein bands were visualized using Coomassie Brilliant Blue (CBB) staining. The Western blotting of the same experiment setup was further used to analyze by anti-AtHYL1 antibody (5:10,000) (AS06136, Agrisera, Vannas, Sweden), according to the report described earlier [37 (link)].

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Bhagat P.K., Verma D., Singh K., Badmi R., Sharma D, & Sinha A.K. (2022). Dynamic Phosphorylation of miRNA Biogenesis Factor HYL1 by MPK3 Involving Nuclear–Cytoplasmic Shuttling and Protein Stability in Arabidopsis. International Journal of Molecular Sciences, 23(7), 3787.

Publication 2022

AS06136

Anti antibody Assay Coomassie brilliant blue Protease Protein Sds page Sensitivity

Corresponding Organization : National Institute of Plant Genome Research

Other organizations : Durham University, Rutgers, The State University of New Jersey, University College Cork

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Variable analysis

independent variables

Type of protein used (AtHYL1FL and AtHYL1N terminal)

dependent variables

Protease sensitivity of the proteins

control variables

SDS-PAGE conditions (12–15% gel)
Protein visualization method (Coomassie Brilliant Blue staining)
Western blotting setup and anti-AtHYL1 antibody (5:10,000) used

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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