Protein extraction was carried out using methods previously described (Mollapour et al., 2010 (link)). Purification was accomplished by two sequential immunoprecipitations. Hsp90α-FLAG for ATPase measurements was expressed in PC3 cells and OGT-FLAG for in vitro GlcNAcylation was expressed in HEK293 cells. Cell lysate was incubated with anti-FLAG antibody conjugated agarose beads (Sigma) for 2hr at 4°C. Immunopellets were washed four times with fresh high salt lysis buffer (20mM Tris-HCl (pH 7.4), 500 mM NaCl, 1 mM MgCl2, 1% NP40, protease inhibitor cocktail (Roche), and PhosSTOP (Roche)) and subsequently competed off with 3x FLAG-peptide (Sigma-Aldrich) in detergent-free fresh lysis buffer (20mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM MgCl2, protease inhibitor cocktail (Roche), and PhosSTOP (Roche)) at 4°C for 1hr two times. Protein was concentrated in 50K Amicon® Ultra Centrifugal Filters (Millipore). A second round of immunoprecipitation, competition and concentration was then performed. Concentrations were determined using the Micro BCA Protein Assay Kit (Thermo Scientific) per manual protocol. Purified protein was run on an SDS-PAGE gel and Coomassie stained to confirm purity prior to use in assays.
50k amicon ultra centrifugal filters
Manufactured by Merck Group
The 50K Amicon® Ultra Centrifugal Filters are a lab equipment product designed for sample concentration and buffer exchange. The filters feature a 50,000 molecular weight cutoff membrane that separates molecules based on size during the centrifugation process.
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2 protocols using 50k amicon ultra centrifugal filters
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Purification of FLAG-tagged Proteins
2
Purification of FLAG-tagged Proteins
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Protein extraction was carried out using methods previously described (Mollapour et al., 2010 (link)). Purification was accomplished by two sequential immunoprecipitations. Hsp90α-FLAG for ATPase measurements was expressed in PC3 cells and OGT-FLAG for in vitro GlcNAcylation was expressed in HEK293 cells. Cell lysate was incubated with anti-FLAG antibody conjugated agarose beads (Sigma) for 2hr at 4°C. Immunopellets were washed four times with fresh high salt lysis buffer (20mM Tris-HCl (pH 7.4), 500 mM NaCl, 1 mM MgCl2, 1% NP40, protease inhibitor cocktail (Roche), and PhosSTOP (Roche)) and subsequently competed off with 3x FLAG-peptide (Sigma-Aldrich) in detergent-free fresh lysis buffer (20mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM MgCl2, protease inhibitor cocktail (Roche), and PhosSTOP (Roche)) at 4°C for 1hr two times. Protein was concentrated in 50K Amicon® Ultra Centrifugal Filters (Millipore). A second round of immunoprecipitation, competition and concentration was then performed. Concentrations were determined using the Micro BCA Protein Assay Kit (Thermo Scientific) per manual protocol. Purified protein was run on an SDS-PAGE gel and Coomassie stained to confirm purity prior to use in assays.
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