Western Blot Analysis of TR146 Cells

TR146 cells were lysed with modified RIPA lysis buffer (32 (link)) containing phosphatase (Sigma-Aldrich) and protease inhibitors (Perbio Science) and separated on 12% SDS-PAGE before being transferred to nitrocellulose membrane. Memrbanes were incubated with primary antibody overnight and developed using the Immobilon ECL reagent (Millipore) ebore being exposed to X-ray film.

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Verma A.H., Zafar H., Ponde N.O., Hepworth O.W., Sihra D., Aggor F.E., Ainscough J.S., Ho J., Richardson J.P., Coleman B.M., Hube B., Stacey M., McGeachy M.J., Naglik J.R., Gaffen S.L, & Moyes D.L. (2018). IL-36 and IL-1/IL-17 drive immunity to oral candidiasis via parallel mechanisms. Journal of immunology (Baltimore, Md. : 1950), 201(2), 627-634.

Publication 2018

Immobilon ECL reagent

Antibody Buffer Cells Immobilon Membrane Nitrocellulose Phosphatase Protease inhibitors Ripa Sds page X ray film

Corresponding Organization : King's College London

Other organizations : University of Leeds, Leibniz-Institut für Naturstoff-Forschung und Infektionsbiologie e. V. - Hans-Knöll-Institut (HKI), Friedrich Schiller University Jena

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Variable analysis

independent variables

Lysis buffer composition (modified RIPA lysis buffer containing phosphatase and protease inhibitors)

dependent variables

Protein expression levels

control variables

SDS-PAGE gel concentration (12%)
Transfer to nitrocellulose membrane
Primary antibody incubation (overnight)

controls

No positive or negative controls were explicitly mentioned in the input protocol.

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