This work was supported by the Department of Education through the Graduate Assistance in Areas of National Need program (P200A100044).

Caution: This protocol requires appropriate training in the use and handling of radioactive materials. Please use the requisite personal protective equipment when performing this assay, including beta radiation shielding. Radioactive waste must be handled carefully, as large volumes of waste are generated during the washing phase of the experiment. Ensure that the waste is stored in an upright container such as a large bucket or bottle that will not be easily knocked over or spilled. Once the experiment is concluded, carefully transfer all l...

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focus+on+phosphohistidine.">Focus on phosphohistidine.</a> Amino acids. 32 (1), 145-156 (2007).</li><li>Kee, J. -. M., Muir, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chasing+phosphohistidine,+an+elusive+sibling+in+the+phosphoamino+acid+family.">Chasing phosphohistidine, an elusive sibling in the phosphoamino acid family.</a> ACS Chem. Biol. 7 (1), 44-51 (2012).</li><li>Tawa, P., Stewart, R. 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The nitrocellulose binding assay we have described has many advantages over previously used methods to characterize histidine kinases. In comparison to traditional SDS/PAGE-based autoradiography, our method is higher throughput and less time-consuming. The nitrocellulose membrane is easier to handle than SDS gels, and does not need to be fixed. Ponceau staining the nitrocellulose allows for the protein spots to be visualized. This provides an easy way to cut out each spot for scintillation counting, and determine that pr...

Adaptive response is crucial for bacterial survival. In order to detect and respond to environmental changes, bacteria use a stimulus-response system known as two-component signaling.1,2 In a typical two-component system, the histidine kinase detects a cognate stimulus, autophosphorylates its conserved histidine residue, then transfers phosphate to a conserved aspartate residue on the receiver domain of a response regulator protein.3 This event triggers a change in the activity of the response regulator, which stimulates a downstream effect.4,5 Thus, bacteria are able to sense and adapt to changes in the local environment. Some two-component signaling systems deviate from this archetype. In some cases, the sensory domain of the histidine kinase is a stand-alone protein, which directly detects the sensory input and modifies kinase activity through a protein-protein interaction.6-8 However, the fundamental process and overall role of the system remains the same. Two-component signaling is a ubiquitous stimulus-response system that is essential for bacterial survival, and histidine kinases play a critical role in the transduction of the signal.9
Despite the importance of histidine kinases to bacterial biology, they remain poorly characterized. This is due to the inherent instability of phosphohistidine, and the lack of a practical method for measuring autophosphorylation. Phosphohistidine is more labile than phosphoserine, phosphothreonine, and phosphotyrosine.10 Thus, techniques that are commonly used to analyze Ser/Thr/Tyr kinases are not applicable for histidine kinases.11 In vitro assays to study histidine kinases have largely been limited to SDS-PAGE autoradiography.12,13 In this method, [&#947;-32P]-ATP is incubated with the kinase, and phosphorylation of the kinase is analyzed by polyacrylamide gel electrophoresis (PAGE) followed by autoradiography of the gel. This method can be used to monitor kinase autophosphorylation, as well as phosphotransfer from the kinase to a response regulator. However, this method has notable shortcomings. PAGE-based assays are low throughput and time-consuming. Such limitations are not conducive to characterizing a protein and ascertaining its kinetic parameters. An alternative method for studying histidine kinases that was published recently utilizes phosphohistidine antibodies to detect autophosphorylation.14 While this method has the advantage of distinguishing between 1-phosphohistidine and 3-phosphohistidine, depending on the instrumentation used for detection, this method may not offer a large dynamic range or high upper limit of detection. Thus, there is a need for a faster, less laborious, and more sensitive assay that can be used to study these important proteins.
Here, we describe and demonstrate a carefully developed nitrocellulose binding assay that can be used to quantify autophosphorylation of purified bacterial histidine kinases in vitro. This assay is higher throughput and less time-consuming than PAGE-based assays. The method also utilizes Cherenkov radiation for phosphohistidine quantification, which offers a high upper limit of detection and a large dynamic range. The assay can be used to determine kinetic parameters for histidine kinases.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Phosphoric acid</td>
			<td>VWR</td>
			<td>AAAA18067-AP</td>
			<td>For quenching reactions and washing nitrocellulose</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>RPI</td>
			<td>T60040-5000.0</td>
			<td>Tris-HCl pH 8.0, for kinase reaction buffer</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>RPI</td>
			<td>P41000-2500.0</td>
			<td>For kinase reaction buffer</td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>RPI</td>
			<td>M24000-500.0</td>
			<td>For kinase reaction buffer</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>RPI</td>
			<td>G22020-4000.0</td>
			<td>For kinase reaction buffer</td>
		</tr>
		<tr>
			<td>5&#39;-ATP</td>
			<td>Promega</td>
			<td>E6011</td>
			<td>Kinase substrate</td>
		</tr>
		<tr>
			<td>[&#947;-32P]-5&#39;-ATP</td>
			<td>Perkin Elmer</td>
			<td>NEG002Z250UC&#160;</td>
			<td>6000 Ci/mmol</td>
		</tr>
		<tr>
			<td>96-well dot blot apparatus</td>
			<td>Bio-rad</td>
			<td>1706545</td>
			<td>For spotting reactions</td>
		</tr>
		<tr>
			<td>Nitrocellulose</td>
			<td>Whatman</td>
			<td>32-10401396-PK</td>
			<td>For spotting reactions</td>
		</tr>
		<tr>
			<td>Ponceau S</td>
			<td>Sigma aldrich</td>
			<td>P3504-50G</td>
			<td>For staining nitrocellulose</td>
		</tr>
	</tbody>
</table>

quantification of bacterial histidine kinase autophosphorylation using a nitrocellulose binding assay

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their involvement in two-component signal transduction, a ubiquitous system through which bacteria sense and respond to environmental stimuli. Two-component signaling features autophosphorylation of a histidine kinase, followed by phosphotransfer to the receiver domain of a response regulator protein, which ultimately leads to an output response. Autophosphorylation of the histidine kinase is responsive to the presence of a cognate environmental stimulus, thereby giving bacteria a means to detect and respond to changes in the environment. Despite their importance in bacterial biology, histidine kinases remain poorly understood due to the inherent lability of phosphohistidine. Conventional methods for studying these proteins, such as SDS-PAGE autoradiography, have significant shortcomings. We have developed a nitrocellulose binding assay that can be used to characterize histidine kinases. The protocol for this assay is simple and easy to execute. Our method is higher throughput, less time-consuming, and offers a greater dynamic range than SDS-PAGE autoradiography.

A representative data set was generated featuring an image captured with a phosphor imager (Figure 1), Ponceau S stain of the nitrocellulose membrane (Figure 2), and scintillation counting data (Figure 3). Figure 3A shows the enzyme kinetic constants in a Lineweaver-Burk plot. These results were obtained using a purified histidine kinase from the gram-negative species Vibrio parahaemolyticus (gene ID 1189383). Th...

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Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay

We report and demonstrate an optimized nitrocellulose binding assay that can be used to quantify autophosphorylation of purified bacterial histidine kinases. Our method has several advantages over traditional SDS-PAGE based techniques, providing a valuable alternative for characterizing these important proteins.

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their ...

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.

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8.4K visualizações.  Stony Brook University. 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 f...