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 liquid waste to appropriately marked radioactive waste containers. Handle all materials with care, and keep a Geiger counter nearby to monitor the workspace for contamination.
NOTE: This protocol is a revised version of a previously reported assay from our group.15 Phosphohistidine stability in H3PO4 should be tested for any uncharacterized histidine kinase prior to using this method. The phosphohistidine stability test has been described previously.15 A negative control that does not contain kinase must be included. This is necessary in order to subtract the background signal from each sample, and ensure that [&#947;-32P]-ATP is sufficiently washed from the membrane.
1. Preparation of Reagents and Materials
<ol>
	<li>Purify histidine kinases from bacterial culture prior to using this assay.
	<ol>
		<li>Purify a histidine kinase (gene ID 1189383) from Vibrio parahaemolyticus (EB101, ATCC 17802) for the development of this assay. Clone the kinase into expression vector pET-23aHis-TEV using NdeI and XhoI restriction sites, and carry out site-directed mutagenesis to yield the mutant construct Vp1876 D499A.</li>
		<li>Transform the plasmid into E. coli BL21 (DE3) pLysS, and grow cells in TB media at 37 &#176;C. Supplement cultures with ampicillin (100 &#181;g/mL) and chloramphenicol (34 &#181;g/mL), and grow with agitation (250 rpm) to an OD600 of 0.6.</li>
		<li>Induce protein expression with IPTG to a final concentration of 25 &#181;M, and grow cultures overnight at 16 &#176;C. Harvest cells by centrifugation (5,000 x g), lyse by sonication, and centrifuge to remove cellular debris (18,000 x g).</li>
		<li>Purify the His-tagged kinase using Ni-NTA agarose. Confirm purity by SDS-PAGE. Optimize purification for each protein, and confirm purity prior to using this assay.</li>
	</ol>
	</li>
	<li>Prepare the 25 mM H3PO4 wash solution. To 1 L of distilled deionized water, add H3PO4 to a final concentration of 25 mM. The pH of this solution is approximately 2.0. Place 25 mM H3PO4 on ice.</li>
	<li>Prepare a 13x stock solution of 325 mM H3PO4, to be used to quench kinase reaction. The pH of this solution is approximately 1.5. Place 325 mM H3PO4 on ice. 
	NOTE: H3PO4 at a final concentration of 25 mM sufficiently quenches the reaction, and also helps to block radiolabeled phosphate that is not bound to histidine from binding to the nitrocellulose membrane.</li>
	<li>Prepare a 4x reaction buffer stock solution containing 160 mM Tris-HCl pH 8.0, 600 mM KCl, 16 mM MgCl2, and 40% glycerol.</li>
	<li>Quantify histidine kinase concentration by established protein concentration methods (Bradford, UV-Vis, etc.).16,17 Prepare a histidine kinase solution with a concentration that is 4 times the desired final concentration. To obtain adequate signal, the final protein concentration in the reaction should ideally be at least 5 &#181;M.</li>
	<li>Prepare a 4x radiolabeled [&#947;-32P]-ATP solution with the appropriate 4x concentration of unlabeled ATP and labeled:unlabeled ratio for the experiment. 
	NOTE: The amount of [&#947;-32P]-ATP that should be included in this mix is dependent on how much [&#947;-32P]-ATP will ultimately be spotted for each reaction. We have obtained adequate signal with final concentrations ranging between 0.1 - 5 &#181;Ci per reaction. It is important that the labeled:unlabeled ATP ratio be kept constant for all reactions in a given experiment. Serial dilutions should be made when multiple ATP concentrations are desired, as this well keep the labeled:unlabeled ATP ratio constant across all concentrations. Final ATP concentrations typically range from 10 &#181;M to at least 1 mM, and each concentration should be assayed in triplicate to obtain reliable kinetic data.</li>
	<li>96- well dot blot apparatus
	<ol>
		<li>Cut an 8 cm x 12 cm piece of nitrocellulose membrane (0.2 &#181;m pore size).</li>
		<li>Position nitrocellulose in a dot blot apparatus, ensuring that the membrane fits such that the apparatus is sealed, and all wells are completely covered by the membrane. If assembled correctly, there should be no air leakage when the vacuum is applied.</li>
		<li>To collect filtrate, connect the apparatus to a secondary side-arm flask. From the valve stem, connect adequate tubing such that the apparatus can be comfortably used without tipping the secondary flask.</li>
		<li>Connect the secondary side-arm flask to aspirator/vacuum source.</li>
		<li>Test that the apparatus is completely sealed by applying 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.</li>
		<li>Optionally, to test the vacuum, pipet 100 &#181;L of reaction buffer into one well. The vacuum should be strong enough to draw all liquid through the well and onto the membrane.</li>
		<li>Turn off vacuum until all samples are ready to be spotted on the membrane.</li>
	</ol>
	</li>
</ol>
2. Reaction Initiation and Quenching
<ol>
	<li>Mixing of reaction components
	<ol>
		<li>Prior to initiation, prepare all four reaction components as 4x stock solutions: reaction buffer (see section 1.4), histidine kinase (1.5), [&#947;-32P]-ATP (1.6), and ddH2O.</li>
		<li>Mix equal volumes of reaction buffer, histidine kinase, and ddH2O. Allow these reaction components to equilibrate at room temperature for a short time (10 min). 
		NOTE: The final reaction volume is dependent on whether the experiment is time-dependent, enzyme-dependent, or substrate-dependent. Time-dependent reactions must be larger, as multiple aliquots are taken from the same reaction and quenched at the desired time point. The reaction volume will depend on the number of time points desired. The assay is optimized for each spot on the nitrocellulose membrane to contain 30 &#181;L of the reaction. Thus, if 10 time points are desired, the reaction volume must be at least 300 &#181;L (a higher volume such as 330 &#181;L would be preferable in the event of any pipetting errors). Enzyme and substrate-dependent experiments require smaller reaction volumes. The reaction volume for these experiments can be as small as 30 &#181;L, as this is the final volume that will be spotted on the nitrocellulose membrane.</li>
		<li>To initiate the reaction, add one volume of [&#947;-32P]-ATP solution to the reaction and mix by pipetting up and down. Track elapsed reaction time with a timer and allow the reaction to proceed for the desired time.</li>
		<li>To quench the reaction, add 1/13 of the total reaction volume of ice-cold 325 mM H3PO4 to the reaction and mix by pipetting up and down. Alternatively, add an aliquot of the reaction to 325 mM H3PO4. 
		NOTE: The final concentration of H3PO4 should be 25 mM to sufficiently quench the reaction. For example, add 2.5 &#181;L 325 mM H3PO4 to 30 &#181;L reaction, or add 30 &#181;L reaction to 2.5 &#181;L 325 mM H3PO4. The final pH of the reaction is approximately 4.0. This concentration of H3PO4 has been tested and confirmed to quench the kinase reaction in this buffer without degrading phosphohistidine.
		<ol>
			<li>For example, mix 7.5 &#181;L 4x reaction buffer, 7.5 &#181;L 4x histidine kinase, and 7.5 &#181;L ddH2O. Initiate reaction with 7.5 &#181;L [&#947;-32P]-ATP. Quench the reaction with 2.5 &#181;L 325 mM H3PO4.</li>
		</ol>
		</li>
		<li>Immediately place quenched reactions on ice until all reactions have been terminated. 
		NOTE: To maximize efficiency, it is advisable to initiate one reaction every 15 or 20 s until all reactions are initiated, and once the desired reaction time has passed, quench one reaction every 15 or 20 s until all are quenched. For those who are using the assay for the first time, a longer interval may be more manageable.</li>
	</ol>
	</li>
</ol>
3. Spotting of Quenched Reactions on Nitrocellulose
<ol>
	<li>Start vacuum on 96-well dot blot apparatus
	<ol>
		<li>Place the pre-assembled 96-well dot blot apparatus (section 1.7) 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.</li>
		<li>Apply vacuum to the 96-well dot blot apparatus.</li>
		<li>Once the apparatus is under vacuum, carefully pipet the quenched reaction directly onto the nitrocellulose membrane in each well. Repeat until all reactions are loaded onto the membrane. Since the binding capacity of nitrocellulose (75 - 110 &#181;g/cm2) is greater than the amount of histidine kinase that is spotted, it can be assumed that nearly all of the kinase binds to the membrane when loaded correctly. 
		NOTE: Depending on the apparatus used, care must be taken not to puncture the nitrocellulose. Observe that all of the reaction has made contact with the nitrocellulose. It is easy for some or all of the reaction to stick to the walls of the well, and not reach the nitrocellulose.</li>
		<li>Wash the wells with 100 &#181;L of ice-cold 25 mM H3PO4. 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.</li>
	</ol>
	</li>
	<li>Apparatus disassembly and nitrocellulose membrane transfer
	<ol>
		<li>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 at this time.</li>
		<li>With forceps, carefully transfer the nitrocellulose membrane from the apparatus to a container of approximately 200 mL 25 mM ice-cold H3PO4. Place a lid on this container, as the wash will be radioactive. At this time, the vacuum may be shut off.</li>
	</ol>
	</li>
</ol>
4. Nitrocellulose Processing
<ol>
	<li>Nitrocellulose washing
	<ol>
		<li>Place the container with the washing membrane on a rocker. Allow the membrane to gently rock for 20 min.</li>
		<li>After 20 min, carefully decant the used wash solution into a large bucket to be used for temporary waste storage. Add 200 mL of ice-cold 25 mM H3PO4 to the membrane and repeat. 
		NOTE: At least three 20 min washes are necessary to remove background [&#947;-32P]-ATP signal from the membrane. After the third wash, test the used wash solution for radiation with a Geiger counter. Continue washing the membrane as described above until no signal is present in the wash solution.</li>
	</ol>
	</li>
	<li>Nitrocellulose drying
	<ol>
		<li>After the membrane is sufficiently washed, allow the membrane to briefly air dry. This typically takes 5 min or less.</li>
	</ol>
	</li>
</ol>
5. Exposure to Storage Phosphor Screen
Note: This section is optional. Exposing the membrane to a phosphor screen is beneficial in that it allows for visualization of the intensity of radiolabeled kinase in each spot on the membrane. The relative intensity of these spots is directly proportional to the amount of phosphorylated histidine kinase in each spot. The intensity can be quantified with image processing software, and these results can be compared to those generated from section 7. Furthermore, this step allows for quality control. Abnormalities seen in this scan might explain irregular results obtained from scintillation counting.
<ol>
	<li>Phosphor screen preparation
	<ol>
		<li>Prior to exposing the membrane to the storage phosphor screen, expose the phosphor screen to white light for at least 5 min. This ensures that any residual image that may be held by the screen is erased.</li>
		<li>Make sure the screen is clean and dry. If necessary, gently clean the screen with a phosphor screen cleaning solution approved by the screen manufacturer, and wipe dry.</li>
	</ol>
	</li>
	<li>Nitrocellulose membrane preparation
	<ol>
		<li>Carefully wrap the dry nitrocellulose membrane in a thin plastic wrap to prevent residual moisture from damaging the phosphor screen. Ensure that there are no wrinkles or bubbles.</li>
	</ol>
	</li>
	<li>Exposure to phosphor screen
	<ol>
		<li>Place the wrapped nitrocellulose membrane in the storage phosphor screen cassette, place the screen face-down onto the membrane, and close the cassette. Do not open the cassette or move the membrane until it is time to scan the phosphor screen, as membrane shifting during exposure will cause a double or smeared image to be captured.</li>
		<li>Expose for at least 4 h, or as long as the phosphor screen manufacturer suggests.</li>
		<li>Once exposure is completed, scan the phosphor screen and capture the image with a phosphor scanner.</li>
	</ol>
	</li>
</ol>
6. Preparing Nitrocellulose Membrane for Scintillation Counting
<ol>
	<li>Ponceau S staining and destaining
	<ol>
		<li>Briefly immerse the nitrocellulose membrane in Ponceau S staining solution (0.1% Ponceau S (w/v) in 5% acetic acid) for 1 - 2 min. Wet the entire membrane with the stain prior to destaining.</li>
		<li>Decant excess Ponceau S from the membrane.</li>
		<li>Rinse the membrane with ddH2O 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. 
		NOTE: This step is necessary to confirm that the kinase is bound to the nitrocellulose, and that protein loading is even throughout all spots. It also allows the spotted kinase to be easily cut out from the membrane.</li>
	</ol>
	</li>
	<li>Cutting out spots
	<ol>
		<li>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 cut out spots will be negligible. It is only important to cut out the entire spot for each reaction.</li>
		<li>Using forceps, carefully transfer each spot into scintillation vials. No scintillation cocktail is necessary as the 32P is readily detectable by Cherenkov radiation. 
		NOTE: Alternatively, the spot can be cut out using a sharpened cork borer. Remove each spot from the nitrocellulose with the cork borer, and push it into a scintillation vial with a pin. With this method, the membrane does not need to be touched, further minimizing radiation exposure.</li>
	</ol>
	</li>
	<li>Determining CPM/pmol of [&#947;-32P]-ATP solution
	<ol>
		<li>Spot several dilutions of [&#947;-32P]-ATP solution (section 1.5) onto 1 cm x 1 cm squares of nitrocellulose. Briefly air dry.</li>
		<li>Using forceps, carefully transfer each square into scintillation vials. No scintillation cocktail is necessary. These samples will be used to generate a standard curve to determine the CPM/pmol of the ATP solution.</li>
	</ol>
	</li>
</ol>
7. Scintillation Counting
<ol>
	<li>Load all scintillation vials into scintillation counter cassettes. Load cassettes into scintillation counter.</li>
	<li>Execute scintillation counting program to measure CPM for each vial. These data, along with the CPM/pmol of the radiolabeled ATP mix (from section 6.3), can be used to calculate the amount of phosphorylated histidine kinase present in each spot, and thus the rate of autophosphorylation.18,19</li>
</ol>

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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 authors have nothing to disclose.

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><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&#039;-ATP</td><td>Promega</td><td>E6011</td><td>Kinase substrate</td></tr><tr><td>[&amp;gamma;-&lt;sup&gt;32&lt;/sup&gt;P]-5&#039;-ATP</td><td>Perkin Elmer</td><td>NEG002Z250UC&amp;nbsp;</td><td>6,000 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...

Watch this Scientific Journal Video about Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay at JoVE.com

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 ...

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Biochemistry

8.5K Views.  Stony Brook University. 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.

Video: Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay

Assaying Protein Kinase Activity with Radiolabeled ATP

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering allosteric details of their regulation. Kinases comprise signaling networks whose defects are often hallmarks of multiple forms of cancer and related diseases, making an assay platform amenable to manipulation of upstream regulatory factors and validation of reaction requirements critical in the search for improved therapeutics. Here, we describe a basic kinase assay that can be easily adapted to suit specific experimental questions including but not limited to testing the effects of biochemical and pharmacological agents, genetic manipulations such as mutation and deletion, as well as cell culture conditions and treatments to probe cell signaling mechanisms. This assay utilizes radiolabeled [&#947;-32P] ATP, which allows for quantitative comparisons and clear visualization of results, and can be modified for use with immunoprecipitated or recombinant kinase, specific or typified substrates, all over a wide range of reaction conditions.

Protein kinases are highly evolved signaling enzymes and scaffolds that are critical for inter- and intracellular signal transduction. We present a protocol for measuring kinase activity through the use of radiolabeled adenosine triphosphate ([&#947;-32P] ATP), a reliable method to aid in elucidation of cellular signaling regulation.

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering ...

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer.
Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin &#945;. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is activated in the G2 phase of the cell cycle and regulates many cellular pathways. Here, we present a protocol for an in vitro kinase assay with Cdk1, which allows the identification of Cdk1-specific phosphorylation sites for establishing cellular targets of this important kinase.

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of fungal secondary metabolites. Inhibitor screening, however, requires purified enzyme activities. Since class 1 deacetylases exert their function as multiprotein complexes, they are usually not active when expressed as single polypeptides in bacteria. Therefore, endogenous complexes need to be isolated, which, when conventional techniques like ion exchange and size exclusion chromatography are applied, is laborious and time consuming. Tandem affinity purification has been developed as a tool to enrich multiprotein complexes from cells and thus turned out to be ideal for the isolation of endogenous enzymes. Here we provide a detailed protocol for the single-step enrichment of active RpdA complexes via the first purification step of C-terminally TAP-tagged RpdA from Aspergillus nidulans. The purified complexes may then be used for the subsequent inhibitor screening applying a deacetylase assay. The protein enrichment together with the enzymatic activity assay can be completed within two days.

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets to treat fungal infections. Here we present a protocol for the specific enrichment of TAP-tagged RpdA combined with an HDAC activity assay that allows in vitro efficacy testing of histone deacetylase inhibitors.

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of ...

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important roles for the cell and may unravel new cellular processes. Among proteins, kinases are major actors as they belong to cell signaling pathways and have the ability to switch on or off many processes crucial to the fate of the cell, such as cell growth, division, differentiation, motility, and death.
In this study, we focused on a new potential kinase protein, LIMK2-1. We demonstrated its existence by Western Blot using a specific antibody. We evaluated its interaction with an upstream regulating protein using coimmunoprecipitation experiments. Coimmunoprecipitation is a very powerful technique able to detect the interaction between two target proteins. It may also be used to detect new partners of a bait protein. The bait protein may be purified either via a tag engineered to its sequence or via an antibody specifically targeting it. These protein complexes may then be separated by SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel) and identified using mass spectrometry. Immunoprecipitated LIMK2-1 was also used to test its kinase activity in vitro by &#947;[32P] ATP labeling. This well-established assay may use many different substrates, and mutated versions of the bait may be used to assess the role of specific residues. The effects of pharmacological agents may also be evaluated since this technique is both highly sensitive and quantitative. Nonetheless, radioactivity handling requires particular caution. Kinase activity may also be assessed with specific antibodies targeting the phospho group of the modified amino acid. These kinds of antibodies are not commercially available for all the phospho modified residues.

We characterized a new kinase protein using robust biochemical approaches: Western Blot analysis with a dedicated specific antibody on different cell lines and tissues, interactions by coimmunoprecipitation experiments, kinase activity detected by Western Blot using a phospho-specific antibody and by &#947;[32P] ATP labeling.

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important ...

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with clinical implications. Several methodologies have been reported to accomplish such screening. However, each has its own limitations (e.g., the screening of only ATP analogues, restriction to using purified kinase domains, significant costs associated with testing more than a few kinases at a time, and lack of flexibility in screening protein kinases with novel mutations). Here, a new protocol that overcomes some of these limitations and can be used for the unbiased screening of kinase inhibitors is presented. A strength of this method is its ability to compare the activity of kinase inhibitors across multiple proteins, either between different kinases or different variants of the same kinase. Self-assembled protein microarrays generated through the expression of protein kinases by a human-based in vitro transcription and translation system (IVTT) are employed. The proteins displayed on the microarray are active, allowing for measurement of the effects of kinase inhibitors. The following procedure describes the protocol steps in detail, from the microarray generation and screening to the data analysis.

A detailed protocol for the generation of self-assembled human protein microarrays for the screening of kinase inhibitors is presented.

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with ...

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of PNKs can be quantified using direct or indirect approaches. Presented here is a direct, in vitro approach to measure PNK activity that relies on a fluorescently-labeled oligonucleotide substrate and polyacrylamide gel electrophoresis. This approach provides resolution of the phosphorylated products while avoiding the use of radiolabeled substrates. The protocol details how to set up the phosphorylation reaction, prepare and run large polyacrylamide gels, and quantify the reaction products. The most technically challenging part of this assay is pouring and running the large polyacrylamide gels; thus, important details to overcome common difficulties are provided. This protocol was optimized for Grc3, a PNK that assembles into an obligate pre-ribosomal RNA processing complex with its binding partner, the Las1 nuclease. However, this protocol can be adapted to measure the activity of other PNK enzymes. Moreover, this assay can also be modified to determine the effects of different components of the reaction, such as the nucleoside triphosphate, metal ions, and oligonucleotides.

This protocol describes a nonradioactive assay to measure kinase activity of polynucleotide kinases (PNKs) on small DNA and RNA substrates.

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of ...

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to measure protein phosphorylation cannot preserve the heterogeneity in phosphorylation across individual proteins. The single-molecule pull-down (SiMPull) assay was developed to investigate the composition of macromolecular complexes via immunoprecipitation of proteins on a glass coverslip followed by single-molecule imaging. The current technique is an adaptation of SiMPull that provides robust quantification of the phosphorylation state of full-length membrane receptors at the single-molecule level. Imaging thousands of individual receptors in this way allows for quantifying protein phosphorylation patterns. The present protocol details the optimized SiMPull procedure, from sample preparation to imaging. Optimization of glass preparation and antibody fixation protocols further enhances data quality. The current protocol provides code for the single-molecule data analysis that calculates the fraction of receptors phosphorylated within a sample. While this work focuses on phosphorylation of the epidermal growth factor receptor (EGFR), the protocol can be generalized to other membrane receptors and cytosolic signaling molecules.

The present protocol describes sample preparation and data analysis to quantify protein phosphorylation using an improved single-molecule pull-down (SiMPull) assay.

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to ...

Development of a Click-Based Assay for Screening HAT1 Enzyme Activity

An Acetyl-Click Chemistry Assay to Measure Histone Acetyltransferase 1 Acetylation

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome assembly. It is required for tumor growth in various systems, making it a potential target for cancer treatment. To facilitate the identification of compounds that can inhibit HAT1 enzymatic activity, we have devised an acetyl-click assay for rapid screening. In this simple assay, we employ recombinant HAT1/Rbap46, which is purified from activated human cells. The method utilizes the acetyl-CoA analog 4-pentynoyl-CoA (4P) in a click-chemistry approach. This involves the enzymatic transfer of an alkyne handle through a HAT1-dependent acylation reaction to a biotinylated H4 N-terminal peptide. The captured peptide is then immobilized on neutravidin plates, followed by click-chemistry functionalization with biotin-azide. Subsequently, streptavidin-peroxidase recruitment is employed to oxidize amplex red, resulting in a quantitative fluorescent output. By introducing chemical inhibitors during the acylation reaction, we can quantify enzymatic inhibition based on a reduction of the fluorescence signal. Importantly, this reaction is scalable, allowing for high throughput screening of potential inhibitors for HAT1 enzymatic activity.

Author Spotlight: Developing Acetyl-Click Assay for HAT1 Inhibitor Screening

Quick and accurate chemical assays to screen for specific inhibitors are an important tool in the drug development arsenal. Here, we present a scalable acetyl-click chemistry assay to measure the inhibition of HAT1 acetylation activity.

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome ...

A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to substrates to create phosphorylated products. The use of &#947;-32-P labeled NTP precursors allows simultaneous monitoring of substrate utilization and product formation by radiography. Thin layer chromatography (TLC) on cellulose plates allows rapid separation and sensitive quantification of substrate and product. We present a method for utilizing the thin-layer chromatography to assay the pyrophosphokinase activity of a purified (p)ppGpp synthetase. This method has previously been used to characterize the activity of cyclic nucleotide and dinucleotide synthetases and is broadly suitable for characterizing the activity of any enzyme that hydrolyzes a nucleotide triphosphate bond or transfers a terminal phosphate from a phosphate donor to another molecule.

A Purification and In Vitro Activity Assay for a pppGpp Synthetase from Clostridium difficile

Here, we describe a method for purifying histidine-tagged pyrophosphokinase enzymes and utilizing thin layer chromatography of radiolabelled substrates and products to assay for the enzymatic activity in vitro. The enzyme activity assay is broadly applicable to any kinase, nucleotide cyclase, or phosphor-transfer reaction whose mechanism includes nucleotide triphosphate hydrolysis.

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to ...

Immunology and Infection

Single-Molecule Pull-Down Assay for Protein Phosphorylation Analysis: A High Throughput Technique to Quantify Protein Phosphorylation in Cell Lysate

Source: Bailey, E. M. et al., <a href="https://www.jove.com/v/63665">An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation.</a> J. Vis. Exp. (2022)
This video demonstrates a sensitive quantification technique of protein phosphorylation using a single-molecule pull-down assay. The functionalization of polyethylene glycol-biotin and the use of labeled antibodies increases the detection of phosphorylated tyrosine with specificity.

Source: Bailey, E. M. et al., An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation. J. Vis. Exp. (2022)
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Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay

Summary

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Chapters in this video

Assaying Protein Kinase Activity with Radiolabeled ATP

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

An Acetyl-Click Chemistry Assay to Measure Histone Acetyltransferase 1 Acetylation

A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile

Single-Molecule Pull-Down Assay for Protein Phosphorylation Analysis: A High Throughput Technique to Quantify Protein Phosphorylation in Cell Lysate