Nitrogenase activity was assessed in vitro by measuring specific activities for H2 and NH3 formation under an N2 or Ar atmosphere. Working under an Ar atmosphere, varying amounts of AnfH2 were dissolved in an anaerobic solution of 50 mM Tris (pH 7.8), 10 mM sodium dithionite, 3.5 mM ATP, 7.87 mM MgCl2, 44.59 mM creatine phosphate and 0.20 mg ml−1 creatine phosphokinase (Sigma-Aldrich, C3755). The reaction vials were sealed by crimping them with butyl rubber stoppers, and the headspace was exchanged to N2 or Ar. Next, the reactions were initialized by adding 0.1 mg of Anf(DGK)2 up to a total volume of 700 µl and were allowed to proceed for 8 min at 30 °C with moderate shaking at 250 r.p.m. Reactions were quenched with 300 µl of 400 mM Na2EDTA solution (pH 8.0), and the amounts of formed H2 and NH3 were analyzed as described below.
C3755
Manufactured by Merck Group
Sourced in United States
The C3755 is a laboratory equipment product manufactured by Merck Group. It is designed for scientific research and analysis applications. The core function of the C3755 is to provide precise and reliable measurements, but a detailed description of its intended use is not available.
Automatically generated - may contain errors
Lab products found in correlation
P7936, by Merck Group (3 mentions)
A2754, by Merck Group (2 mentions)
Creatine phosphokinase, by Merck Group (1 mentions)
Proteinase k, by Thermo Fisher Scientific (1 mentions)
Rnase t1, by Thermo Fisher Scientific (1 mentions)
Phosphorimager, by GE Healthcare (1 mentions)
Rnasin, by Promega (1 mentions)
A7699, by Merck Group (1 mentions)
Phosphocreatine, by Merck Group (1 mentions)
Nocodazole, by Merck Group (1 mentions)
S blebbistatin, by LGC (1 mentions)
M0250, by Merck Group (1 mentions)
Mgcl2, by Merck Group (1 mentions)
Kh2po4, by Merck Group (1 mentions)
6 protocols using c3755
1
Nitrogenase Activity Assay in Vitro
2
In vitro Transcription Assay Protocol
3
Preparation of Thioester and Isopeptide Ubiquitin Conjugates
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To prepare thioester E2~Ub conjugates for assays, WT UbcH5b (100-200 μM) was incubated with E1 (1 μM) and ubiquitin (300-600 μM) at 37 °C for 40 minutes in 20 mM HEPES pH7.5, 50 mM NaCl, 2 mM MgCl2, 2 mM TCEP (C4706, Sigma-Aldrich) and 10 mM ATP (A7699, Sigma-Aldrich). The reaction products were subsequently separated using a SuperdexTM 75 10/300 GL column equilibrated in 20 mM HEPES pH 7.5, 150 mM NaCl. The eluted conjugate was analysed by SDS-PAGE, and the fractions corresponding to the E2~Ub conjugate were pooled and concentrated to ~100 μM before snap-freezing.
Isopeptide linked UbcH5b~Ub conjugate for crystallography and crosslinking purposes was prepared using wild-type ubiquitin and UbcH5b, which included mutations to allow isopeptide conjugate formation (C85K), prevent backside binding (S22R) and increase E2 stability (C21S, C107S, C111S). The reaction contained E1 (1 μM), E2 (100–200 μM), ubiquitin (300–600 μM) and ATP (4 mM) (A7699, Sigma-Aldrich) in cycling buffer [25 mM Tris pH 9.5, 2.5 mM MgCl2, 5 mM phosphocreatine (P7936, Sigma-Aldrich) and 0.6 U/mL creatine phosphokinase (C3755, Sigma-Aldrich)]. Typically a 5 mL reaction was incubated at 37 °C for 18-24 hours then separated on a HiLoadTM 26/600 SuperdexTM 75 prep column, pre-equilibrated in 20 mM Tris-HCl pH 7.5, 200 mM NaCl47 (link).
Isopeptide linked UbcH5b~Ub conjugate for crystallography and crosslinking purposes was prepared using wild-type ubiquitin and UbcH5b, which included mutations to allow isopeptide conjugate formation (C85K), prevent backside binding (S22R) and increase E2 stability (C21S, C107S, C111S). The reaction contained E1 (1 μM), E2 (100–200 μM), ubiquitin (300–600 μM) and ATP (4 mM) (A7699, Sigma-Aldrich) in cycling buffer [25 mM Tris pH 9.5, 2.5 mM MgCl2, 5 mM phosphocreatine (P7936, Sigma-Aldrich) and 0.6 U/mL creatine phosphokinase (C3755, Sigma-Aldrich)]. Typically a 5 mL reaction was incubated at 37 °C for 18-24 hours then separated on a HiLoadTM 26/600 SuperdexTM 75 prep column, pre-equilibrated in 20 mM Tris-HCl pH 7.5, 200 mM NaCl47 (link).
4
Xenopus Egg Extract Replication Assay
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HSS and NPE were prepared as described previously (24 (link)) and stored at −80°C. Before both bulk- and single-molecule replication assays, each 33-μl aliquot of HSS was supplemented with 250 ng of nocodazole (Sigma-Aldrich, M1404) and 1 μl of an adenosine triphosphate (ATP) regeneration system, containing 650 mM phosphocreatine (Sigma-Aldrich, P7936), 65 mM ATP (pH 7.0; Sigma-Aldrich, A2754) and creatine phosphokinase (0.161 mg/ml; Sigma-Aldrich, C3755). Similarly, each 16-μl aliquot of NPE was supplemented with 0.5 μl of ATP mix. Activated extracts were centrifuged for 5 min at 16,000g, room temperature and used in replication assays. All Xenopus work fully complied with the UK Animals (Scientific Procedures) Act 1986 as implemented by the Francis Crick Institute.
5
Relaxing and Activating Solutions for Muscle Fiber Experiments
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The composition of the solutions was as described [24 (link)]. The relaxing solution contained 80 mM K-propionate, 20 mM imidazole, 10 mM EGTA, 4 mM ATP, 5 mM MgCl2, 20 mM phosphocreatine and 300 U/ml creatine phosphokinase (C3755, Sigma-Aldrich, USA) (pH=7.2). In the pre-activating solution, the concentration of EGTA was reduced to 0.2 mM. The activating solution contained 10.1 mM CaCl2 in addition to the components of the relaxing solution. BTS was obtained from Sigma Rare Chemicals (USA) or Tokyo Chemical Industry Co. (Japan), and (S)-(−)-blebbistatin was obtained from Toronto Research Chemicals (Canada) or Calbiochem (USA). BDM was obtained from Sigma-Aldrich. When necessary, these chemicals were added to the three solutions described above.
6
Enzymatic Assays for Creatine and Creatine Phosphate
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The assays for creatine and creatine phosphate were based on the enzymatic reactions:
Creatine and creatine phosphate were measured by the consumption of NADPH measured by absorption at 340 nm at 37°C. Extracted sample was suspended in a working solution (200 mM phosphate buffer [200 mM KH2PO4 (P5504; Sigma) and 200 mM Na2HPO4 (4062-01; JT Baker, Phillipsburg, NJ)], 7 mM MgCl2 (M0250; Sigma), 1.8 mg NADPH (Calzyme), 10 mM of α-ketoglutarate (75892; Sigma), and 2.5 mM ADP (A2754; Sigma) and the reactions were catalyzed by adding 6 U of glutamate dehydrogenase (GLDH; G4387; Sigma), 100 U of urease (U1875; Sigma), and 90 U of creatinase (C2409; Sigma). After creatine is consumed, creatine phosphate was measured by adding 42 U creatine kinase (CK; C3755; Sigma).
Creatine and creatine phosphate were measured by the consumption of NADPH measured by absorption at 340 nm at 37°C. Extracted sample was suspended in a working solution (200 mM phosphate buffer [200 mM KH2PO4 (P5504; Sigma) and 200 mM Na2HPO4 (4062-01; JT Baker, Phillipsburg, NJ)], 7 mM MgCl2 (M0250; Sigma), 1.8 mg NADPH (Calzyme), 10 mM of α-ketoglutarate (75892; Sigma), and 2.5 mM ADP (A2754; Sigma) and the reactions were catalyzed by adding 6 U of glutamate dehydrogenase (GLDH; G4387; Sigma), 100 U of urease (U1875; Sigma), and 90 U of creatinase (C2409; Sigma). After creatine is consumed, creatine phosphate was measured by adding 42 U creatine kinase (CK; C3755; Sigma).
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