According to the manufacturer’s instruction, cell lysates were subjected to lambda PPase (New England Biolabs) treatment. Control reactions lacking the phosphatase were performed in parallel. The assay mixture was then used to perform the co-IP assay, as described above.
Lambda ppase
Manufactured by New England Biolabs
Lambda PPase is a phosphatase enzyme that removes phosphate groups from serine, threonine, and tyrosine residues in proteins. It can be used to dephosphorylate proteins for various applications in protein research and analysis.
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Lab products found in correlation
2 protocols using lambda ppase
1
Phosphatase Treatment for Co-IP
2
Protoplast-based Immunoblot and Dual Reporter Assays
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Arabidopsis protoplasts isolated from the leaves of 5-week-old plants were transfected with the constructs as indicated (Zhang et al., 2010a) . Twelve hours after transfection, protoplasts were harvested and used for experiments. For immunoblot analyses, protoplasts were treated with or without PAMPs as indicated for 5 min. Total protein was extracted with extraction buffer (50 mM HEPES [pH 7.5], 150 mM KCl, 1 mM EDTA, 1 mM DTT, 1% Triton X-100, 1×complete protease inhibitors) and subjected to anti-FLAG (Sigma F3165, 1:10,000 dilution) or anti-HA (Roche 11666606001, 1:5,000 dilution) immunoblot. For PPase treatment, total protein was treated with lambda PPase (New England Biolabs) according to manufacturer's instructions. For dual reporter analyses, the protoplasts were incubated overnight under faint light and LUC activity was recorded by a microplate luminometer (Berthhold Centro XS 3 ).
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