Thin Layer Chromatography Based Enzyme Activity Assay: A Technique to Determine In Vitro Pyrophosphokinase Activity Using Thin Layer Chromatography

1. Protein Activity Assay by Thin Layer Chromatography

1. Prepare the thin layer chromatography (TLC) plate.
   1. 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.
   2. 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. Washing the plates in two perpendicular directions further ensures that any contaminants present in the resin are isolated in one corner of the plate (Figure 1).
   3. Bring the plates out of the glass chamber and leave on a benchtop rack to dry overnight (12–18 h).
   4. Mark the dry plates 2.0 cm from one edge with a soft pencil to indicate where the samples will be applied for TLC. For 2 μL samples, apply samples no less than 1.0 cm apart (Figure 1).
      NOTE: This allows clear separation between adjacent spot, which is critical for signal quantification. As long as the cellulose resin is not scratched, small pencil marks on the surface will not interfere with solvent migration.
   5. When planning experiments, always leave one spot on each plate unused.
      NOTE: This will provide a blank lane for sample quantification (Figure 1). A 20 cm TLC plate will have room for 19 spots.
2. Enzyme activity assay
   1. Prepare a 5x buffer mix containing 50 mM Tris-HCl (pH 7.5), 25 mM ammonium acetate, 10 mM KCl, 1 mM DTT and 0.6 mM ATP (adenosine triphosphate).
      NOTE: This mix may be prepared in large quantities and frozen in 10 μL aliquots for later use. Do not subject mix to multiple freeze-thaw cycles.
   2. Prepare individual reactions containing 3 μM RSH (Rel/Spo homolog enzymes), 1x buffer mix, 0.6 mM GDP (guanosine diphosphate), 1.2 mM MgCl₂. Add 1.0 μCi of γ-³²P-ATP (radiolabelled ATP) per 10 μL of reaction and use nuclease free water to bring the reaction up to a desired volume. Add the RSH after the other components have been mixed, as the addition of RSH to the nucleotide-containing mix initiates the enzymatic activity assay.
      NOTE: Final reaction volume will depend on the number of timepoints sampled. To sample 2 μL/timepoint, assemble 10 μL of reaction mixture for each 4 timepoints.
   3. To control ATP hydrolysis from contaminating nuclease activity, assemble a 10 μL reaction containing no protein and incubate it in parallel. Spot 2 μL samples at t = 0 and at the end of the experiment to ensure that ATP was not hydrolyzed in the absence of protein.
   4. Immediately upon addition of RSH, remove 2 μL and spot it onto the labelled PEI-cellulose plate as the t = 0 min sample.
   5. Incubate the reaction at 37 °C, removing 2 μL aliquots at desired timepoints.
      NOTE: Enzymatic activity will cease when the sample is adsorbed onto the cellulose plate. Wait 10–30 min after the last spot is added to plate before development to ensure complete adsorption and sample drying.
3. Thin layer chromatography
   1. Fill the chromatography chamber with 1.5 M 1.5 M KH₂PO₄ (pH 3.64) to a depth of 0.5 cm.
      NOTE: The volume needed will depend on the dimensions of the chromatography tank. Any glass container with a level bottom that is wide enough to allow insertion of the TLC plate without bending may be used as a developing tank with the addition of a cover. The TLC plate can be cut into narrower strips with a clean razorblade to enable development in a glass beaker covered in plastic film.
   2. Immerse the bottom edge of the plate in solvent. Allow the solvent to migrate to the top of the plate (~90 min).
      NOTE: While solvent migration will halt at the top of the plate and samples will not be lost or run together during a longer immersion, plates should not be left in solvent overnight. Immersions longer than 4 h can cause the resin to detach from the plate backing and result in loss of signal.
   3. Remove the plate from the chromatography tank and place it on a benchtop drying rack.
   4. Allow the plate to air dry overnight.
      NOTE: Drying may be accelerated using a hair drier. Dryness can be assessed by the color of the resin, which will darken when wet and return to the color of an unused plate when completely dry.
   5. After the plate is dry, wrap the plate in plastic film to avoid transfer of radioactive material to the imaging cassette and analyze by autoradiography (Figure 2).
4. Data analysis
   1. Expose the PEI-cellulose plate containing separated reactions to a phosphorimager cassette for 4 h at room temperature.
      NOTE: This is sufficient exposure to yield a very clear image using the indicated concentrations of fresh γ-³²P-ATP. If lower amounts of radiolabelled substrate are used, exposure time can be increased to 12-16 h.
   2. Image the cassette on a phosphorimager.
   3. Using imaging software with a graphical user interface, draw Regions of Interest (ROIs) by selecting Draw Rectangle and using the mouse to draw rectangular ROIs around one entire lane and the ATP and ppGpp (Guanosine tetraphosphate) spots contained within that lane (Figure 2).
   4. Use the Select, Copy, and Paste commands (or corresponding commands based on the imaging software being used) to draw identical ROIs within the other lanes to ensure that the ROIs are measuring signal within identical areas in each lane. Include ROIs from an unused lane, to be used as blanks.
   5. Using the Analyze | Tools | ROI Manager | Add commands of the imaging software, select all of the ROIs drawn on the PEI cellulose plate.
   6. Using the Analyze | Set Measurements | Measure commands, quantify the signal intensity within each ROI and export the measurements as a spreadsheet (Figure 2). Subtract blank ROI values from experimental signals.
      
   7. Calculate what percentage of the blanked signal within each lane is attributable to ATP and ppGpp using the formulas.
      NOTE: ROIs may be drawn and quantitated using commercial software compatible with the phosphorimager or freely available ImageJ software (National Institutes of Health).