Protein Extraction and Analysis from Mouse Epidermis

Cultured keratinocytes were lysed in RIPA buffer containing 1× HALT phosphatase/protease inhibitors (Invitrogen, Waltham, MA) and PMSF (Sigma-Aldrich, St. Louis, MO) and briefly sonicated on ice as described (29 (link)). For epidermal lysates, frozen mouse skin was scraped to remove the epidermal layer, which was then ground using a frozen mortar and pestle, placed in skin lysis buffer containing 1× HALT and PMSF and processed as described (26 ). Protein concentrations for epidermal samples were determined using the Dc protein assay (BioRad, Hercules, CA). Samples were separated on SDS-PAGE, transferred to PVDF membranes and processed using established protocols (26 ). Protein bands were visualized using Amersham ECL reagents (GE Healthcare Life Sciences, Pittsburgh, PA). Results are representative of n≥3 for cell culture studies; 3 mice/group for mouse epidermis studies which were repeated at least two times.

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Janda J., Burkett N., Blohm-Mangone K., Huang V., Curiel-Lewandrowski C., Alberts D.S., Petricoin EF I.I.I., Calvert V.S., Einspahr J., Dong Z., Bode A.M., Wondrak G.T, & Dickinson S.E. (2016). Resatorvid-based Pharmacological Antagonism of Cutaneous TLR4 Blocks UV-induced NF-κB and AP-1 Signaling in Keratinocytes and Mouse Skin. Photochemistry and photobiology, 92(6), 816-825.

Publication 2016

HALT phosphatase/protease inhibitors PMSF Dc protein assay Amersham ECL reagents

Assay Buffer Cell culture Epidermis Frozen Keratinocytes Membranes Mice mouse Mouse Phosphatase Protease inhibitors Protein Pvdf Ripa Sds page Skin

Corresponding Organization : University of Arizona

Other organizations : George Mason University, University of Minnesota

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Variable analysis

independent variables

Cell culture treatment or mouse epidermis sample type (e.g., wild-type vs. genetically modified)

dependent variables

Protein expression levels or protein modifications

control variables

Lysis buffer composition (RIPA buffer, 1× HALT phosphatase/protease inhibitors, PMSF)
Sonication procedure for cell lysis
Scraping and grinding procedure for mouse epidermis samples
Skin lysis buffer composition (1× HALT, PMSF)
Protein quantification method (DC protein assay)
SDS-PAGE separation and PVDF membrane transfer
Immunoblotting and visualization (Amersham ECL reagents)

controls

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

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