As previously published [30 (link)], the relaxing solution contained 4 mM Mg-ATP, 1 mM free Mg2+, 10–6.00 mM free Ca2+, 20 mM imidazole, 7 mM EGTA, 14.5 mM creatine phosphate and KCl to adjust the ionic strength to 180 mM and pH to 7.0. Additionally, the rigor buffer for Mant-ATP chase experiments contained 120 mM K acetate, 5 mM Mg acetate, 2.5 mM K2HPO4, 50 mM MOPS, 2 mM DTT with a pH of 6.8. The lambda phosphatase solution (New England Biolabs) was prepared by a 100-fold dilution into the relaxing solution to yield 4 U lambda phosphatase/µl [29 (link)]. The solution for extracting myosin regulatory light chains (RLC) contained 20 mM EDTA, 50 mM KPr, 10 mM potassium phosphate buffer with a pH of 7.1 [5 (link)]. Finally, the solution for extracting myosin-binding protein C (MyBP-C) contained 10 mM EDTA, 31 mM Na2HPO2, 124 mM NaH2PO4, with a pH of 5.9 [7 (link), 8 (link)].
Lambda phosphatase solution
Manufactured by New England Biolabs
Lambda phosphatase solution is a recombinant enzyme that catalyzes the hydrolysis of phosphate groups from a wide range of phosphorylated proteins and peptides. It is a highly active and efficient enzyme used in various biochemical and molecular biology applications.
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Lab products found in correlation
2 protocols using lambda phosphatase solution
1
Preparation of Myosin Regulatory and Binding Proteins
2
Skeletal Muscle Fiber Permeabilization
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Relaxing solution for permeabilized skeletal muscle fibers contained 1 mM DTT, 100 mM KCl, 10 mM imidazole, 2.0 mM EGTA, 4.0 mM ATP, and 1 mM (free, 5 total) MgCl2. Minimal Ca2+ activating solution (pCa 9.0) for experimental protocol contained 7.00 mM EGTA, 20 mM imidazole, 5.42 mM MgCl2, 72.37 mM KCl, 0.016 mM CaCl2, 14.50 mM PCr, and 4.7 mM ATP. Maximal Ca2+ activating solution (pCa 4.5) for experimental protocol contained 7.00 mM EGTA, 20 mM imidazole, 5.26 mM MgCl2, 60.25 mM KCl, 7.01 mM CaCl2, 14.50 mM PCr, and 4.81 mM ATP. A range of Ca2+ concentrations for experiments was prepared by varying combinations of maximal and minimal Ca2+ solutions. Preactivating solution contained 0.5 mM EGTA, 20 mM imidazole, 5.42 mM MgCl2, 98.18 mM KCl, 0.016 mM CaCl2, 14.50 mM PCr, and 4.8 mM ATP. PKA solution was prepared by diluting 1 mg DTT in 100 µl of ultrapure water; the DTT solution was then added to 400 U PKA (Sigma), and the 100 µl PKA was then diluted in 700 µl pCa 9.0 solution to yield 0.5 U PKA/µl. Lambda phosphatase solution (New England Biolabs) was prepared by a 100-fold dilution into pCa 9.0 solution to yield 4 U lambda phosphatase/µl.
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