Western Blot Protein Detection

Total protein extraction was performed as previously described (Barco et al., 2019b (link)). Extract [5 µl (DEX : MYB51-myc or DEX : WRKY33-flag) or 50 µl (DEX : MYB51-myc and DEX : WRKY33-flag)] was loaded onto a 10% SDS-PAGE gel, and the separated proteins were transferred to PVDF membrane (Millipore, Billerica, MA), stained with Ponceau S for labeling of total protein, and probed with either FLAG M2 (Sigma-Aldrich, cat# F1804) or c-Myc 9E10 (Santa Cruz Biotechnology, cat# sc-40) antibodies diluted 1:1,000 in 1X PBS containing 5% (w/v) nonfat milk.

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Barco B, & Clay N.K. (2020). Hierarchical and Dynamic Regulation of Defense-Responsive Specialized Metabolism by WRKY and MYB Transcription Factors. Frontiers in Plant Science, 10, 1775.

Publication 2019

PVDF membrane FLAG M2 c-Myc 9E10

Antibodies C myc Membrane Milk Ponceau s Protein Pvdf Sds page

Corresponding Organization : Yale University

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Variable analysis

independent variables

DEX : MYB51-myc
DEX : WRKY33-flag

dependent variables

Total protein extraction
Protein separation and transfer
Protein detection using FLAG M2 or c-Myc 9E10 antibodies

control variables

10% SDS-PAGE gel
PVDF membrane (Millipore, Billerica, MA)
Ponceau S staining for total protein labeling
Antibody dilution (1:1,000) in 1X PBS containing 5% (w/v) nonfat milk

controls

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

Annotations

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