Thin Layer Chromatography Based Enzyme Activity Assay: A Technique to Determine In Vitro Pyrophosphokinase Activity Using Thin Layer Chromatography
Published: April 30, 2023
Abstract
Source: Pokhrel, A., et al. A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile. J. Vis. Exp. (2018).
This video demonstrates a thin-layer chromatography-based enzyme assay to determine pyrophosphokinase activity using radiolabeled nucleotides. The method provides insights into nucleotide synthesis and phosphotransfer reactions.
Protocol
1. Protein Activity Assay by Thin Layer Chromatography Prepare the thin layer chromatography (TLC) plate. Prepare polyethyleneimine (PEI)-cellulose plates by washing in deionized water. Place the plates in a glass chamber with double distilled water to a depth of ~0.5 cm. Allow water to migrate to the top of the plate. NOTE: Washing the plates is not strictly necessary, as plates may be used without it, but washing does increase the clarity of resulting images. …
Representative Results
Figure 1: Preparation of TLC plates. (A) Plates are washed in one dimension by placing the bottom edge in water (blue). Contaminants (yellow) migrate to the top of the plate with the solvent. (B) After a plate is dried completely, it is washed in a second dimension by rotating it 90° relative to the first wash and again allowing water to migrate to the top of the plate. (C) After washing, any …
Divulgaciones
The authors have nothing to disclose.
Materials
| Thin layer chromatograph (TLC) development tank | General Glass Blowing Company | 80-3 | |
| Polyethylenimine (PEI)-cellulose plates (20 cm x 20 cm, 100 μm thickness) with polyester support | Sigma-Aldrich | Z122882-25EA | |
| ATP, [γ-32P]- 3000 Ci/mmol 10mCi/ml lead, 100 μCi | Perkin Elmer | NEG002A | |
| Adenosine 5’-triphosphate (ATP) 100 mM | Bio Basic Canada | AB0311 | |
| Guanosine-5’-diphosphate disodium salt (GDP) | Alfa Aesar | AAJ61646MC/E | |
| Storage phosphor screen | GE Healthcare Life Sciences | BAS-IP TR 2040 E Tritium Screen | |
| Storm 860 phosphorimager | GE Healthcare Life Sciences | – |
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Thin Layer Chromatography Based Enzyme Activity Assay: A Technique to Determine In Vitro Pyrophosphokinase Activity Using Thin Layer Chromatography. J. Vis. Exp. (Pending Publication), e21103, doi: (2023).