In vitro γ-secretase assays using the recombinant APP-C100-Flag substrate and purified γ-secretase were performed as previously described (Cacquevel et al., 2008 (link) and Fraering et al., 2005 (link)). Briefly, γ-secretase solubilized in 0.2% (wt/vol) CHAPSO-HEPES (pH 7.5) was incubated at 37°C for 4 hours with 1 mM APP-C100-Flag substrate, 0.1% (wt/vol) PC, 0.025% PE and 10 mM of bithionol, hexachlorophene or mitoxantrone. All compounds were added to the reactions from DMSO stock solutions before the addition of APP-C100Flag. After 4 hours of incubation at 37°C, the reactions were halted by adding Laemmli sample buffer for western blot analyses. The resulting products, APP intracellular domain (AICD)-Flag and Aβ, were detected with a Flag-specific M2 antibody (1:1000; Sigma-Aldrich) and an Aβ–specific 6E10 antibody (1:1000; Covance), respectively (Cacquevel et al., 2008 (link) and Fraering et al., 2005 (link)).
Flag specific m2 antibody
Manufactured by Merck Group
The Flag-specific M2 antibody is a laboratory tool used for the detection and identification of proteins tagged with the Flag peptide sequence. The antibody specifically binds to the Flag epitope, which is a short amino acid sequence commonly used to label recombinant proteins. This antibody can be utilized in various immunological techniques, such as Western blotting, immunoprecipitation, and immunofluorescence, to study the expression, localization, and interactions of Flag-tagged proteins.
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2 protocols using flag specific m2 antibody
1
In vitro γ-secretase Assay
2
Quantification of γ-Secretase Activity Using Recombinant Substrates
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γ-Secretase assays using the recombinant human Notch and Amyloid precursor protein substrates Notch100-Flag and APP-C100-Flag were performed as previously reported61 (link)62 (link)63 (link). Membrane proteins were extracted from Ptenpc+/+- and Ptenpc−/−-null mice prostate samples in 50 mM HEPES (pH 7.0) with 1% CHAPSO. The protein content in the extracts was normalized by BCA and incubated in 0.2% (wt/vol) CHAPSO, 50 mM HEPES (pH 7.0), 150 mM NaCl, 5 mM MgCl2 and 5 mM CaCl2 and incubated at 37 °C for 4 h with 1 μm substrate, 0.1% (wt/vol) phosphatidylcholine and 0.025% (wt/vol) phosphatidylethanolamine. The generated products AICD (Amyloid Intracellular C-terminal Domain)-Flag and NICD (Notch Intracellular Domain)-Flag were analysed by WB and detected with Flag-specific M2 antibody (Sigma-Aldrich).
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