Pupal Wing Western Blotting Protocol

For pupal wing westerns, 28-30 hr APF pupal wings were dissected directly into sample buffer. Two pupal wing equivalents were loaded per lane. Westerns were probed with affinity-purified rat anti-Pk [49 (link)], affinity purified rabbit anti-Dsh [49 (link)], rabbit anti-Stbm [50 (link)] and monoclonal mouse anti-actin (AC-40, Sigma, UK). A BioRad ChemiDoc XRS+ was used for imaging. Images were quantified using the Gel Analysis plug-in in ImageJ.

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Warrington S.J., Strutt H., Fisher K.H, & Strutt D. (2017). A Dual Function for Prickle in Regulating Frizzled Stability during Feedback-Dependent Amplification of Planar Polarity. Current Biology, 27(18), 2784-2797.e3.

Publication 2017

AC-40 ChemiDoc XRS+

Actin Buffer Mouse Pupal Rabbit

Corresponding Organization : University of Sheffield

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Variable analysis

independent variables

No independent variables explicitly mentioned

dependent variables

Protein levels of Pk, Dsh, and Stbm

control variables

Pupal stage (28-30 hr APF)
Tissue (pupal wings)
Protein loading (two pupal wing equivalents per lane)
Antibodies used (affinity-purified rat anti-Pk, affinity purified rabbit anti-Dsh, rabbit anti-Stbm, monoclonal mouse anti-actin)
Imaging method (BioRad ChemiDoc XRS+)
Image quantification method (Gel Analysis plug-in in ImageJ)

controls

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

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