The overall goal of this protocol is to quantify autophosphorylation of purified bacterial histidine kinases. This method can help answer key questions in the field of two-component signaling. Such as the effect of cognate stimuli in protein-protein interactions on autophosphorylation of histidine kinases.
The main advantages of this technique are that it's a high throughput method that can be used to accurately detect picomoles of phosphohistadine formed and obtain kinetic parameters for histidine kinases. As a post-doctoral fellow, I use filtration on nitrocellulose membranes to quantify protein-bound radioactive ligands. Consequently, it made sense to develop a similar technique for quantifying histidine phosphorylation in bacterial histidine kinases.
Begin this procedure with preparation of reagents and materials as described in the text protocol. To set up the 96 well dot-plot apparatus, cut an eight centimeter by 12 centimeter piece of nitrocellulose membrane. Position the nitrocellulose in the dot-plot apparatus.
Ensure that the membrane fits such that the apparatus is sealed and all wells are completely covered by the membrane. To collect the filtrate, connect the apparatus to a secondary sidearm flask. From the valve stem, connect adequate tubing to allow the apparatus to be comfortably used without tipping the secondary flask.
Then, connect the secondary sidearm flask to an aspirator vacuum source. Test that the apparatus is completely sealed by applying a vacuum to the apparatus. If the apparatus is correctly assembled, a strong vacuum will be present in all of the wells.
All tubing connections can be wrapped in Parafilm or Saran wrap to ensure a tight seal. Prior to initiation, prepare all four reaction components as four X stock solutions, as described in the text protocol. Mix equal volumes of reaction buffer, histidine kinase, and double distilled water.
Allow these reaction components to equilibrate at room temperature for a short time. To initiate the reaction, add one volume of gamma-32 P ATP solution and mix by pippetting up and down. Track the elapsed reaction time with a timer and allow the reaction to proceed for the desired time.
Quench the reaction. Add an aliquot of the reaction to 325 millimolar phosphoric acid. Immediately place the quenched reactions on ice until all reactions have been terminated.
Place the preassembled 96 well dot-plot apparatus into a secondary container. This container should be large enough for the apparatus to be easily disassembled in, as the apparatus and membrane will be radioactive after use. Apply a vacuum to the 96 well dot-plot apparatus.
Once the apparatus is under vacuum, carefully pipette the quenched reaction directly onto the nitrocellulose membrane in each well. Repeat this process until all reactions are loaded onto the membrane. Since the binding capacity of nitrocellulose is greater than the amount of spotted histidine kinase, it can be assumed that nearly all of the kinase binds to the membrane when loaded correctly.
Depending on the apparatus used, take care not to puncture the nitrocellulose. Observe that all of the reaction has made contact with the nitrocellulose. It is easy for some of the reaction to stick to the walls of the well.
Wash the wells with 100 microliters of ice cold 25 millimolar phosphoric acid. This will allow any reaction volume that may have been trapped in the well to reach the membrane, ensuring complete loading of all reactions. Allow the entire volume to flow through the membrane.
Carefully disassemble the apparatus before shutting off the vacuum. Be advised that both the apparatus and nitrocellulose membrane are radioactive at this time. Do not remove any apparatus components from the secondary container.
With forceps, carefully transfer the nitrocellulose membrane from the apparatus to a container of approximately 200 milliliters of 25 millimolar ice cold phosphoric acid. Place a lid on this container, as the wash will be radioactive. At this time, the vacuum may be shut off.
Place the container with the washing membrane on a rocker. Allow the membrane to gently rock for 20 minutes. After 20 minutes, carefully decant the used wash solution into a large bucket to be used for temporary waste storage.
Add 200 milliliters of ice cold 25 millimolar phosphoric acid to the membrane. Wash the membrane in this manner at least three times to remove background gamma-32 P ATP signal from the membrane. After the third wash, test the used wash solution for radiation with a Geiger counter.
Continue to wash the membrane until no signal is present in the wash solution. After the membrane is sufficiently washed, allow the membrane to briefly air dry, which typically takes five minutes or less. After exposure of the nitrocellulose to a phosphorus green, as described in the text protocol, prepare for Ponceau S staining and destaining.
Briefly immerse the nitrocellulose membrane in Ponceau S staining solution for one to two minutes. Wet the entire membrane with the stain prior to destaining. Then, decant the excess Ponceau S from the membrane.
Rinse the membrane with double distilled water until only the spots from the histidine kinase are stained. Note that this procedure will only work if all spots contain detectable amounts of protein. Using scissors, cut out each spot on the nitrocellulose membrane.
It is not necessary to cut perfectly along the edge of the stained spot. If the membrane was washed sufficiently, background due to varying size of cutout spots is negligible. It is only important to cut out the entire spot for each reaction.
Using forceps, carefully transfer each spot into scintillation vials. No scintillation cocktail is necessary, as the phosphorus 32 is readily detectable by Cherenkov radiation. Next, spot several dilutions of gamma-32 P ATP solution onto one centimeter by one centimeter squares of nitrocellulose.
Using forceps, carefully transfer each square into scintillation vials sans scintillation cocktail. These samples will be used to generate a standard curve to determine the counts per minute per picomole of the ATP solution. Ponceau staining of the nitrocellulose membrane is shown here.
Staining is uniform throughout all samples, indicating that all samples are equally loaded. Fluorography results of the nitrocellulose membrane show the relative level of radiolabeled phosphohistidine in each spot. The fourth and eighth row are negative controls demonstrating that excess ATP is washed from the nitrocellulose membrane.
Representative results of quantification of histidine kinase autophosphorylation are shown here. This data is generated from scintillation counting of each spot cut out from the membrane. The standard curve of counts per minute per picomole of ATP shown here is also generated from scintillation counting and is used to calculate the picomoles of phosphohistidine present in each sample.
Following this procedure, other methods, like introducing cognate stimuli or proteins that are hypothesized to modulate kinase activity, can be added into the experiment to answer additional questions like the effect of these additives on kinase activity. Don't forget that working with radioactivity can be extremely hazardous. Precautions such as proper radiation safety training and the use of personal protective equipment and shielding should always be made while performing this procedure.
