Filaggrin Detection in Skin Samples

Western blot analysis of skin for filaggrin detection was performed as previously described (10 (link)). In brief, epidermis was separated from dermis following incubation in PBS with 5 mM EDTA at 56°C. Epidermis was homogenized in protein extraction buffer (50 mM Tris, 8 M Urea, pH 7.6 with HALT protease inhibitor cocktail (Thermo Scientific)) using an Ultra-Turrax. Lysates were clarified by centrifugation (13,000 g, 4°C for 15 min). Proteins were resolved by SDS-PAGE and transferred onto a PVDF membrane (Thermo Scientific). Membrane-bound proteins were incubated with primary rabbit anti-mouse filaggrin antibody (Covance) and secondary HRP-conjugated goat anti-rabbit immunoglobulins (Dako). Enhanced chemiluminescence substrate (Thermo Scientific) was used to visualise membrane bound filaggrin.

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Floudas A., Saunders S.P., Moran T., Schwartz C., Hams E., Fitzgerald D.C., Johnston J.A., Ogg G.S., McKenzie A.N., Walsh P.T, & Fallon P.G. (2017). Interleukin-17 receptor A maintains and protects the skin barrier to prevent allergic skin inflammation. Journal of immunology (Baltimore, Md. : 1950), 199(2), 707-717.

Publication 2017

HALT protease inhibitor cocktail Ultra-Turrax PVDF membrane HRP-conjugated goat anti-rabbit immunoglobulins Enhanced chemiluminescence substrate

Anti immunoglobulins Buffer Centrifugation Chemiluminescence Dermis Edta Epidermis Filaggrin Goat Membrane Membrane proteins Mouse Protease inhibitor 7 Proteins Pvdf Rabbit Sds page Skin Tris Urea Western blot analysis

Corresponding Organization : National Children’s Research Centre

Other organizations : Queen's University Belfast, Amgen (United States), Medical Research Council, National Institute for Health Research, University of Oxford, MRC Laboratory of Molecular Biology

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Variable analysis

independent variables

Separation of epidermis from dermis following incubation in PBS with 5 mM EDTA at 56°C

dependent variables

Filaggrin detection by Western blot analysis

control variables

Protein extraction buffer (50 mM Tris, 8 M Urea, pH 7.6 with HALT protease inhibitor cocktail (Thermo Scientific))
SDS-PAGE and transfer onto a PVDF membrane (Thermo Scientific)
Primary rabbit anti-mouse filaggrin antibody (Covance)
Secondary HRP-conjugated goat anti-rabbit immunoglobulins (Dako)
Enhanced chemiluminescence substrate (Thermo Scientific)

controls

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

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