Pei cellulose f

To measure AMPylation‐/deAMPylation‐dependent AMP production by FICD proteins, reactions were set up in HKM buffer containing 250 μM ATP, 0.0185 MBq [α‐³²P]‐ATP, 3 mM TCEP, 5 μM ATP hydrolysis‐deficient BiP^T229A (UK 838) and 2 μM FICD variant in a final volume of 30 μl. The reactions were started by addition of nucleotides and incubated for 2 h at 30°C. Afterwards, 2 μl was spotted onto a thin‐layer chromatography (TLC) plate (PEI Cellulose F; Merck Millipore) pre‐spotted with 2 μl of nucleotide mix containing AMP, ADP and ATP (each at 3.5 mM). The TLC plate was developed with 400 mM LiCl and 10% (v/v) acetic acid as a mobile phase and the dried plates were exposed to a storage phosphor screen. The signals were detected with a Typhoon biomolecular imager and quantified using ImageJ64.

RecG_Nm ATP hydrolysis activity was monitored by thin-layer chromatography (TLC), as previously described [7 (link)]. RecG_Nm or RecG_NmK294A was added to initiate a 10 μl reaction in the presence of 100nM DNA cofactor in ATPase buffer [20 mM Tris/HCl (pH 7.5), 2 mM MgCl₂, 100 μg BSA/ml, 25 mM cold ATP, 0.023 nM [γ-³²P]ATP, 2 mM DTT]. Also reactions containing DNA cofactor but without the wild type (RecG_Nm) protein, and RecG_Nm but without DNA cofactor were included per experiment. The reaction mixture was incubated at 37°C for the indicated times and terminated by adding 5 μl of 0.5 M EDTA (pH 8.0). Samples (2 μl) were spotted onto TLC plates (PEI Cellulose F, Merck) at 1.5 cm intervals and resolved using a solution containing 1 M formic acid and 0.5 M LiCl. The TLC plates were air-dried, exposed to a phosphorimaging screen, imaged and quantified as described above for the DNA binding assays. The percentage of hydrolyzed ATP was calculated as {counts for γ-32Pi / (counts for γ-32Pi+counts for [γ-32P]ATP)} x 100. The values obtained from samples lacking RecG_Nm were subtracted from the samples containing RecG_Nm to account for background ATP hydrolysis.