Activated CD4+ T cells transduced with FoxP3 or mutants (1×109 cells/sample) were lysed in NP-40 lysis butter (20mM Tris-HCl, 2mM EDTA, 1% NP-40 10% glycerol, 150mM NaCl, 1mM PMSF and 1X Proteinase inhibitor cocktail; Roche, #04693116001) for 2 hr with gentle vortexing at 4°C. The lysates were sedimented at 15,000 rpm for 30 min at 4°C to remove aggregates, and the supernatant injected into an FPLC Superose 6 column (10 X 300mm, GE Healthcare Life Science) at a flow rate of 400ul/min in PBS. Twenty seven fractions of 400 ul were collected, immediately concentrated with filter centrifugation (Centricon 10,000 KDa cutoff, Millipore) down to 50ul, resolved by SDS-PAGE and probed by sequential immunoblotting for FoxP3 (eBioscience, #14-5773). FoxP3 containing fractions were incubated with anti-RELA (Abcam, #ab7970) or anti-EZH2 (Active motif, #AC22), revolved by SDS-PAGE and immunoblotted with anti-FoxP3.
Centricon 10 000 kda cutoff
Manufactured by Merck Group
The Centricon 10,000 KDa cutoff is a lab equipment product designed for separating and concentrating macromolecules, such as proteins and enzymes, from solutions. It utilizes a membrane with a molecular weight cutoff of 10,000 Daltons to selectively retain molecules above that size while allowing smaller molecules to pass through.
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Lab products found in correlation
2 protocols using centricon 10 000 kda cutoff
1
Analysis of FoxP3 Interactions
2
Analysis of FoxP3 Interactions
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