When calf intestine phosphatase (CIAP) (New England Biolabs: NEB) was used, Xenopus embryos overexpressing HA-tagged Xl_Nemp1 (Xl_Nemp1-HA) were lysed in lysis buffer A. Lysates were incubated with anti-HA antibody at 4 °C for 1 h, then added with protein G-agarose beads, and incubated for another 1.5 h. The beads were washed 3 times with lysis buffer A, once with NEBuffer 3 (NEB), and incubated in NEBuffer 3 containing 0.5 u/ml of CIAP for 3 h at room temperature. When λ protein phosphatase (NEB) was used, Xenopus embryos overexpressing mouse Nemp1-HA (Mm_Nemp1-HA) were lysed in lysis buffer A without EDTA. Lysates were incubated with λ protein phosphatase in NEBuffer for Protein MetalloPhosphatases (NEB) for 45 min at 30 °C. Treated samples were analyzed by western blotting with anti-HA antibody.
Nebuffer for protein metallophosphatases
Manufactured by New England Biolabs
NEBuffer for Protein MetalloPhosphatases is a buffer solution designed for the optimal activity of protein metalloPhosphatases. It provides the necessary ionic conditions and pH range to support the enzymatic function of these phosphatases.
Automatically generated - may contain errors
2 protocols using nebuffer for protein metallophosphatases
1
Dephosphorylation of Nemp1 Proteins
2
Atg14-FLAG Dephosphorylation Assay
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Cells expressing Atg14-FLAG were treated with rapamycin for 2 h. Atg14-FLAG in the cell lysates was then bound to anti-FLAG antibody-conjugated beads as described in the methods for immunoprecipitation. The beads were washed three times with NEBuffer for Protein MetalloPhosphatases (New England Biolabs, Inc.) and incubated with lambda protein phosphatase (New England Biolabs, Inc.) with or without heat inactivation (at 65°C for 1 h) at 30°C for 1 h. The beads were then washed with IP Buffer and the bound proteins were eluted by incubating the beads in SDS sample buffer at 65°C for 10 min. After removal of the beads, DTT was added to the eluates to a final concentration of 20 mM, followed by immunoblotting analysis.
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