Assays for reconstitution of SAM-ClbH formation (50 μL) contained 50 mM Tris-HCl (pH 8.3), 200 mM NaCl, 10 mM MgCl2, 1 mM TCEP, 250 μM CoA, 3 μM ClbH, ClbH-C-A2-PCP or ClbH-C-A2-PCP S1524A, 0.6 μM Sfp, 3 mM ATP, and 25 μM [carboxyl-14C]-SAM (55 mCi/mmol, American Radiolabeled Chemicals). ClbH, ClbH-C-A2-PCP or ClbH-C-A2-PCP S1524A was incubated with Sfp and CoA at 22 °C for 1.5 h for phosphopantetheinylation. The complete reaction was initiated by the addition of [carboxyl-14C]-SAM and ATP. The assay mixtures were incubated at 30 °C and quenched after 1 h by the addition of 100 μL BSA (1 mg/mL) and 500 μL trichloroacetic acid (10% m/v). The samples were centrifuged (10,000 rpm × 8 min), and the supernatant was removed. The protein pellet was washed twice with 250 μL trichloroacetic acid (10% m/v), re-suspended in 100 μL formic acid, and added to 10 mL of scintillation fluid (Ultima Gold, Perkin Elmer). Radioactivity was measured on a Beckman LS 6000SC scintillation counter.
Carboxyl 14c sam
Manufactured by American Radiolabeled Chemicals
[carboxyl-14C]-SAM is a radiolabeled compound that contains a carbon-14 isotope at the carboxyl group of S-adenosyl methionine (SAM). It is a useful research tool for investigating biological processes involving the transfer of methyl groups.
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2 protocols using carboxyl 14c sam
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Assay for SAM-ClbH Complex Formation
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Assay for SAM-ClbH Complex Formation
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Assays for reconstitution of SAM-ClbH formation (50 μL) contained 50 mM Tris-HCl (pH 8.3), 200 mM NaCl, 10 mM MgCl2, 1 mM TCEP, 250 μM CoA, 3 μM ClbH, ClbH-C-A2-PCP or ClbH-C-A2-PCP S1524A, 0.6 μM Sfp, 3 mM ATP, and 25 μM [carboxyl-14C]-SAM (55 mCi/mmol, American Radiolabeled Chemicals). ClbH, ClbH-C-A2-PCP or ClbH-C-A2-PCP S1524A was incubated with Sfp and CoA at 22 °C for 1.5 h for phosphopantetheinylation. The complete reaction was initiated by the addition of [carboxyl-14C]-SAM and ATP. The assay mixtures were incubated at 30 °C and quenched after 1 h by the addition of 100 μL BSA (1 mg/mL) and 500 μL trichloroacetic acid (10% m/v). The samples were centrifuged (10,000 rpm × 8 min), and the supernatant was removed. The protein pellet was washed twice with 250 μL trichloroacetic acid (10% m/v), re-suspended in 100 μL formic acid, and added to 10 mL of scintillation fluid (Ultima Gold, Perkin Elmer). Radioactivity was measured on a Beckman LS 6000SC scintillation counter.
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