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2 protocols using rabbit polyclonal anti flag

1

Antibody Validation and Protein Detection

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Rabbit polyclonal anti-Flag, anti-GFP, anti-HA, and anti-GPS2, and mouse monoclonal anti-actin and anti-SUMO1 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-Myc and mouse monoclonal anti-Flag (M2) antibodies were purchased from Sigma-Aldrich (St. Louis, MO). Cycloheximide was obtained from Sigma-Aldrich, and MG132 was obtained from Calbiochem (San Diego, CA). Clean Blot IP Detection Reagent was purchased from Thermo Scientific (Rockford, IL).
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2

Detecting ALS-linked Proteins by Western Blot

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mouse monoclonal anti-GFP at ½000 (cat. no sc-9996, Santa Cruz Biotechnology), mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (cat. no G8795, Gillingham), and mouse monoclonal histone H3 (cat. no 96C10, New England Biolabs) were used for detecting lysate, soluble, and insoluble fractions on Western blot from NP-40 insolubility assays. In the annexin A11/EF-hand protein (calcyclin, ALG-2, and sorcin) binding assays, mouse monoclonal anti-FLAG (cat. no. F3165, Sigma-Aldrich) was used to immunoprecipitate FLAG-tagged EF-hand proteins, and mouse monoclonal anti-GFP (cat. no. sc-9996, Santa Cruz Biotechnology) and mouse monoclonal anti-FLAG or rabbit polyclonal anti-FLAG (cat. no. F7425, Sigma-Aldrich) were used to detect GFP-fused proteins and FLAG-tagged proteins, respectively. Polyclonal rabbit anti-ANXA11 (cat. no. 10479–2-AP, Proteintech) was used for ANXA11R235Q staining of HEK cells (fig. S4) and spinal cord of the p.D40G SALS patient, SALS patient devoid of known ALS causing mutation, other neurodegenerative disorders, and controls. Polyclonal rabbit anticalcyclin (cat. no. 10245–1-AP, Proteintech) was used for immunohistochemistry (IHC) of patient and control postmortem tissue and detection of FLAG-tagged calcyclin by Western blot in HEK cells.
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