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Protocol for the Bottom-Up Proteomic Analysis of Mouse Spleen.

STAR protocols (2020-12-31)
Paul Dowling, Stephen Gargan, Margit Zweyer, Michael Henry, Paula Meleady, Dieter Swandulla, Kay Ohlendieck
ABSTRACT

This protocol describes the comparative proteomic profiling of the spleen of wild type versus mdx-4cv mouse, a model of dystrophinopathy. We detail sample preparation for bottom-up proteomic mass spectrometry experiments, including homogenization of tissue, protein concentration measurements, protein digestion, and removal of interfering chemicals. We then describe the steps for mass spectrometric analysis and bioinformatic evaluation. For complete details on the use and execution of this protocol, please refer to Dowling et al. (2020).

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Trizma® base, Primary Standard and Buffer, ≥99.9% (titration), crystalline
Sigma-Aldrich
Sodium dodecyl sulfate, BioReagent, suitable for electrophoresis, for molecular biology, ≥98.5% (GC)
Sigma-Aldrich
Ammonium bicarbonate, ReagentPlus®, ≥99.0%
Sigma-Aldrich
Acetonitrile, suitable for HPLC, gradient grade, ≥99.9%
Sigma-Aldrich
Trifluoroacetic acid, ReagentPlus®, 99%