Mid-log phase cells were lysed with 10% TCA and resuspended with 2× SDS sample buffer. The dilution buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 µM EDTA, and 0.5% Tween-20) was added to the lysate and incubated with 10,000 U/µl PPase (New England Biolabs, Inc.) and 150 U/ml TEVp (Promega) at 30°C for 1 h. Reactions were terminated with equal volumes of 2× SDS sample buffer.
Ppase
Manufactured by New England Biolabs
Sourced in China
PPase is a laboratory product manufactured by New England Biolabs. It functions as a pyrophosphatase, catalyzing the hydrolysis of pyrophosphate to inorganic phosphate.
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Lab products found in correlation
4 protocols using ppase
1
Protein Phosphatase Assay Protocol
2
Enzymatic DNA Phosphorylation Protocol
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PPase (New England Biolabs, Beijing, China, Cat. No. M2403S) was directly added to the DNA mixture after Pfu extension. After 30 min of incubation at RT, the samples were used for Pi measurements.
3
Pex14p Phosphorylation Analysis
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Pex14p TPA affinity-purified from Triton X-100 extracts was mixed with 10x phosphatase buffer (0.5 M HEPES, 1 M NaCl, 20 mM DTT, 0.1% Brij 35, pH 7.5), MnCl2 (1 mM final concentration), and 800 units of lambda protein phosphatase (-PPase; New England Biolabs) and incubated for 20 min at 30°C under slight agitation. As control, the reaction was performed without -PPase using the same amount of Pex14p TPA . Reactions were stopped by adding SDS sample buffer and proteins were separated by Phos-tag SDS-PAGE using a standard 10% SDS polyacrylamide gel additionally containing 50 µM Phos-tag reagent (Phos-tag acrylamide AAL-107; Wako Pure Chemical Industries, Ltd., Japan) and 100 mM MnCl2 in the separation gel. Electrophoresis was carried out at 80 V for 4 h. Subsequently, the gel was incubated for 20 min in Western blot transfer buffer (25 mM Tris, 192 mM glycine, 20% [v/v] methanol) containing 10 mM EDTA to quench the Phos-tag reagent and washed for 10 min in transfer buffer without EDTA. Proteins were transferred to a polyvinylidene difluoride membrane at a constant voltage of 30 V and 4°C for 14 h.
4
Phosphorylation of PER2 by CK1
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Complexes of PER2 and wild-type or constitutively active CK1 were allowed to form for 30 min at 25°C before adding kinase buffer [30 mM Hepes (pH 7.5), 7 mM MgCl 2 , 0.5 mM DTT, 100 M ATP, and protease inhibitor cocktail] in a final volume of 20 l. Reactions were maintained for 30 min at 37°C and terminated by the addition of Laemmli buffer. When indicated, PPase (New England Biolabs) was added directly to the substrate (PER2) and reactions were allowed to proceed for 30 min at 30°C before they were terminated. Depending on the experiment, proteins were resolved on 4% (for PER2) and 8% [for CK1 and CK1(1-305)] SDS-PAGE or Phos-tag (WACO Chemicals, USA) gels and visualized by immunoblotting.
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