We thank Dr. Andrew Sikkema and Andrea Kaminski for their critical reading of this manuscript. This work was supported by the US National Institute of Health Intramural Research Program; US National Institute of Environmental Health Sciences (NIEHS; ZIA ES103247 to R.E.S) and the Canadian Institutes of Health Research (CIHR; 146626 to M.C.P).

1. Preparation
<ol>
	<li>Prepare buffer and reagents.
	<ol>
		<li>Make 1x Reaction Buffer by combining 20 &#181;L of 1 M Tris (pH = 8.0), 40 &#181;L of 5 M sodium chloride, 2.5 &#181;L of 2 M magnesium chloride, 100 &#181;L of 50% (v/v) glycerol, and RNase-free water to reach a total volume of 1 mL.</li>
		<li>Make urea loading dye by combining 4.8 g of urea, 200 &#181;L of 1 M Tris (pH = 8.0), 20 &#181;L of 0.5 M EDTA (pH = 8.0), 0.5 mL of 1% (w/v) bromophenol blue, and RNase-free water to reach a total volume of 10 mL.</li>
		<li>Make 10x TBE buffer by combining 108 g of Tris base, 55 g of boric acid, 40 mL of 0.5 M EDTA (pH = 8.0), and RNase-free water to reach a total volume of 1 L.</li>
		<li>Make 10% (w/v) ammonium persulfate (APS). Weigh 0.5 g of APS and add 3 mL of RNase-free water to dissolve the solid. Add RNase-free water to reach a total volume of 5 mL. Wrap the tube in foil and store the solution at 4 &#176;C. 
		NOTE: Dissolved APS decays over time. Replace the 10% APS stock every 2 weeks.</li>
	</ol>
	</li>
	<li>Obtain polynucleotide kinase enzyme.
	<ol>
		<li>Acquire pure Las1-Grc3 PNK enzyme2 stored in Reaction Buffer (see step 1.1.1) in a stock concentration of 20 &#956;M. The storage buffer will vary depending on the specific PNK.</li>
	</ol>
	</li>
	<li>Prepare nucleic acid substrate. 
	NOTE: This protocol monitors 5&#39;-phosphorylation of a 27 nt RNA substrate that harbors the Las1-Grc3 processing site (C2 site) contained within the Saccharomyces cerevisiae pre-ribosomal internal transcribed spacer 2 (SC-ITS2; see Table 1)2,25,26.
	<ol>
		<li>Chemically synthesize an RNA oligonucleotide substrate containing a 5&#39;-hydroxyl end along with a 3&#39;-fluorescent label. The fluorophore will be used for the visualization of the RNA. Ensure that the fluorophore is positioned on the RNA end that is not targeted for phosphorylation. As an alternative to commercial sources, internal and terminal fluorescent labeling of RNA substrates can be performed in-house using cost-effective protocols27.</li>
		<li>Remove excess fluorophore from the RNA labeling reaction by HPLC-purification28.</li>
		<li>Resuspend the lyophilized RNA using RNase-free water to 500 nM.</li>
		<li>Store RNA aliquots at -80 &#176;C for long-term storage or -20 &#176;C for short-term storage.</li>
	</ol>
	</li>
	<li>Prepare the ATP concentration series.
	<ol>
		<li>Make a 20 mM working stock of ATP using Reaction Buffer (see step 1.1.1). Use this working stock to generate four serial dilutions ranging from 20 mM-0.02 mM.</li>
		<li>To make the first dilution of the concentration series, mix 1 &#956;L of the 20 mM ATP working stock with 9 &#956;L of Reaction Buffer. This will result in a 10-fold dilution of the ATP with a final concentration of 2 mM.</li>
		<li>Use the 2 mM ATP stock generated in step 1.4.2 to make the next dilution in the concentration series. Mix 1 &#956;L of the 2 mM ATP stock with 9 &#956;L of Reaction Buffer. This will make a new ATP stock concentration of 0.2 mM.</li>
		<li>Continue diluting 1 &#956;L of the previous ATP stock with 9 &#956;L of Reaction Buffer to make the subsequent ATP stock in the concentration series.</li>
	</ol>
	</li>
</ol>
2. In vitro RNA kinase reaction
NOTE: This assay can be used to measure several variables such as time, nucleotide levels, or enzyme concentration. The goal of this experiment is to assess the amount of phosphorylated RNA in the presence of constant Las1-Grc3 complex and varying ATP levels.
<ol>
	<li>For each RNA-enzyme kinase reaction, combine 1 &#956;L of 500 nM RNA substrate, 8.3 &#956;L of 130 nM Las1-Grc3, and 0.2 &#956;L of 5 mM EDTA. 
	NOTE: If carrying out several reactions, prepare a master stock of the RNA-enzyme mixture and aliquot 9.5 &#956;L of this master mix to each reaction tube.</li>
	<li>Start the assay.
	<ol>
		<li>Set the heat block to 37 &#176;C.</li>
		<li>At 10 s intervals, mix 0.5 &#956;L of one ATP substock from the ATP concentration series prepared in step 1.4 with one RNA-enzyme mixture and place the reaction in the 37 &#176;C heat block. 
		NOTE: Add 0.5 &#956;L of Reaction Buffer instead of ATP to one RNA-enzyme mixture for a PNK negative control. Use another necessary control as well (i.e., RNA substrate in the absence of PNK enzyme).</li>
		<li>Incubate the reactions for 60 min at 37 &#176;C. 
		NOTE: Fluorescently-labeled RNA is light sensitive; therefore, cover the reactions with foil.</li>
		<li>Using the same order as in step 2.2.2, quench each reaction every 10 s by spiking the reaction with 10 &#956;L of urea loading dye (see step 1.1.2). 
		NOTE: In addition to 4 M urea, proteinase K may be added at this step to degrade the enzyme. Follow the instructions provided by the supplier to determine the required protease amount and reaction conditions.</li>
		<li>Use the quenched in vitro RNA kinase reactions immediately for downstream analysis (see section 3) or store at -20 &#176;C to be analyzed at a later date.</li>
	</ol>
	</li>
</ol>
3. Gel electrophoresis
<ol>
	<li>Prepare 15% acrylamide/8 M urea gel solution. 
	CAUTION: Acrylamide must be handled with care, because it is a neurotoxin. Always wear gloves, a lab coat, and goggles when handling it.
	<ol>
		<li>In a 150 mL glass beaker, combine 22.5 mL of premixed 40% acrylamide/bis-acrylamide 29:1 solution, 6 mL of 10x TBE (see step 1.1.3), 28.8 g of urea, and RNase-free water to a total volume of 59 mL. Gently stir the solution. 
		NOTE: Depending on the RNA substrate length, altering the percentage of polyacrylamide may improve the resolution between unphosphorylated and phosphorylated RNA.</li>
		<li>To dissolve the urea, heat the solution in the microwave for 20 s, stir the liquid, and immediately place the solution back into the microwave for another 20 s. Gently stir the solution until the urea completely dissolves.</li>
		<li>Slowly cool the solution by placing the glass beaker into a shallow water bath containing cold water. Make sure that the level of cold water surrounding the glass beaker is above the level of solution inside the glass beaker. This will promote efficient heat transfer. Wait 5 min. 
		NOTE: Do not continue with this protocol if the glass beaker feels warm; the water should be below 25 &#176;C. If it still feels warm after 5 min, then replace the water in the water bath with fresh cold water and wait another 5 min.</li>
		<li>Filter and degas the solution using a 0.22 &#956;m disposable filtration unit to remove particulates and microscopic air bubbles.</li>
	</ol>
	</li>
	<li>Pour the denaturing gel.
	<ol>
		<li>Use soap and warm water to clean a short and long glass plate designed for gels with overall dimensions of 31.0 cm x 38.5 cm. Spray each glass plate with 95% ethanol and wipe the glass to remove any moisture. 
		NOTE: One of the two plates can be siliconized to prevent damage to the gel when separating the glass plate sandwich. However, this step is not necessary because this protocol is designed to visualize the RNA without separating the glass plates.</li>
		<li>Place the long glass plate horizontally on top of a box so it is elevated off the benchtop. 
		CAUTION: Acrylamide is toxic, therefore the gel pouring station must be covered in bench paper that can absorb any spilled liquid and be immediately placed in a waste bag after the procedure is complete.</li>
		<li>Place a clean 0.4 mm spacer along the long edges of the long glass plate.</li>
		<li>Lay the short glass plate atop the long plate and ensure the edges of the short plate, long plate, and spacers are aligned. Clamp each side using three evenly spaced metal clamps.</li>
		<li>Add 24 &#956;L of TEMED to the 15% acrylamide/8 M urea solution prepared in step 3.1 and mix the solution.</li>
		<li>Add 600 &#956;L of 10% (w/v) APS (see step 1.1.4) to the solution in step 3.2.5 and gently mix the solution.</li>
		<li>Immediately pour the solution between the glass plates. 
		NOTE: To avoid bubbles, tap the glass as the solution is poured.</li>
		<li>Carefully add a clean, 0.4 mm, 32 well comb to the top of the glass plate sandwich.</li>
		<li>Allow a minimum of 30 min for the acrylamide to polymerize.</li>
	</ol>
	</li>
	<li>Run the denaturing gel.
	<ol>
		<li>Set the heat block to 75 &#176;C.</li>
		<li>Remove the metal clamps holding the glass plate sandwich together and thoroughly wash and dry the glass plate sandwich.</li>
		<li>Place the glass plate sandwich into the gel apparatus with the short plate facing inward.</li>
		<li>Prepare 0.5x TBE running buffer by combining 100 mL of 10x TBE with 1.9 L RNase-free water. Add 600 mL of the running buffer to the upper and lower chambers of the gel apparatus.</li>
		<li>Gently remove the comb and rinse the wells thoroughly using a syringe. 
		NOTE: This step is critical for removing urea from the wells.</li>
		<li>Pre-run the gel for 30 min at 50 W. Voltage is approximately 2,000 V. 
		CAUTION: This gel apparatus operates at a high wattage and users should exhibit precaution.</li>
		<li>Rinse the wells thoroughly using a syringe. 
		NOTE: This step is critical for even loading of sample into the wells.</li>
		<li>Pulse spin the quenched reactions from step 2.2.5. Then incubate the tubes at 75 &#176;C for 3 min. Repeat the pulse spin.</li>
		<li>Immediately load 10 &#956;L of sample per well and run the gel for 3 h at 50 W. 
		NOTE: Fluorescently-labeled RNA is light sensitive; therefore, cover the gel apparatus with foil.</li>
	</ol>
	</li>
	<li>Image the denaturing gel.
	<ol>
		<li>Turn off the power supply and drain the upper chamber of the gel apparatus.</li>
		<li>Wash and dry the outer side of the glass plate sandwich using soap and water. Cover the glass plate sandwich with foil while transporting the gel.</li>
		<li>Mount the glass plate sandwich onto the stage of a laser scanner capable of quantitative and sensitive fluorescence detection. 
		NOTE: This gel is extremely thin for maximum resolution. For this reason, this protocol is designed to avoid the difficulties of removing the gel from the glass plate sandwich by directly visualizing the fluorescently-labeled RNA through the glass plates. The use of commercially available low-fluorescence glass plates will increase the captured signal by improving the signal-to-noise ratio.</li>
		<li>Set the excitation and emission wavelengths of the laser scanner for the desired fluorophore. 
		NOTE: The optimal excitation and emission wavelengths may differ for specific fluorophores. For the FAM fluorophore, the excitation and emission wavelengths are 495 nm and 535 nm, respectively.</li>
		<li>Define the area to be visualized on the laser scanner stage and image the gel according to the manufacturer&#39;s instructions (Figure 1).</li>
	</ol>
	</li>
</ol>
4. Image analysis and signal quantification
<ol>
	<li>Load the digital gel image acquired in step 3.4.5 into image procesing software (see Table of Materials).</li>
	<li>Using the left button of the mouse, click and drag a rectangle to define a template box to mark the boundaries on the digital gel image that will be used to quantify each RNA band. The RNA band seen in the the reaction mixture set in the absence of PNK enzyme is usually a good band to generate this template. 
	NOTE: To avoid bias caused by background signal, it is critical that the area surrounding each RNA band being measured is identical. For this reason, it is best to use the same template box to demarcate each RNA band.</li>
	<li>Open the ROI Manager under &#34;Tools&#34; and mark the position of the template box by clicking &#34;Add&#34;. 
	NOTE: Quantitation programs can also draw boxes automatically following the software user manual.</li>
	<li>Using the left button of the mouse, click and drag the template box over to the next RNA band and repeat step 4.3. Continute this process until the position of all the unphosphorylated and phosphorylated RNA bands in each reaction have been marked.</li>
	<li>Measure the integrated density within each box by clicking &#34;Measure&#34; in the ROI Manager.</li>
	<li>To calculate the relative amount of phosphorylated RNA in a reaction, divide the integrated density of the phosphorylated RNA band by the sum of the integrated densities of the unphosphorylated and phosphorylated RNA bands from the same reaction. This approach can also be applied to calculate the relative amount of unphosphorylated RNA.</li>
	<li>To visualize the accumulation of phosphorylated RNA product and the corresponding depletion of unphosphorylated RNA substrate across the ATP concentration series, plot the relative amount of RNA calculated in step 4.6 against the concentration of ATP.</li>
</ol>

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The authors have nothing to disclose.

Described is an assay to measure kinase activity of Grc3 PNK on fluorescently-labeled nucleotide substrates. This protocol can be applied to characterize other PNK enzymes by adapting the Reaction Buffer and oligonucleotide substrate. For instance, the protocol calls for a trace amount of EDTA. The addition of EDTA is beneficial for two reasons: First, this approach favors magnesium-bound Grc3 by preventing the enzyme from binding to trace amounts of contaminating metals in the mixture. Second, a small amount of EDTA inh...

Polynucleotide kinases (PNK) play critical roles in many DNA and RNA processing pathways, such as DNA repair and ribosome assembly1,2,3,4,5. These fundamental enzymes catalyze the transfer of the terminal (gamma) monophosphate from a nucleoside triphosphate (NTP, most often ATP) to the 5&#39; hydroxyl end of a nucleotide substrate. One of the most well characterized PNKs is bacteriophage T4 PNK, which has broad substrate specificity and is heavily utilized by molecular biology labs for incorporating radioactive isotope labels onto the 5 terminus of a DNA or RNA substrate6,7,8,9,10,11,12. Another example of a PNK enzyme is CLP1, which is found in Eukarya, Eubacteria, and Archaea, and is implicated in several RNA processing pathways4,13,14,15.
Historically, most assays that measure polynucleotide kinase activity are dependent upon radioactive isotope labeling and subsequent autoradiography5,16. In recent years a number of additional assays have been developed to measure PNK activity, including single molecule approaches, microchip electrophoresis, molecular beacons, as well as colorimetric and luminescence-based assays17,18,19,20,21,22. While many of these new approaches provide enhanced detection limits and avoid the use of radioactivity, each has drawbacks, such as cost, reliance on immobilized resin, and limitations in substrate choice.
Grc3 is a polynucleotide kinase that plays a pivotal role in the processing of pre-ribosomal RNA2,3,23. Grc3 forms an obligate complex with the endoribonuclease Las1, which cleaves the Internal Transcribed Spacer 2 (ITS2) of the pre-ribosomal RNA3. Cleavage of the ITS2 by Las1 generates a product harboring a 5&#39; hydroxyl that is subsequently phosphorylated by the Grc3 kinase3. To investigate the nucleotide and substrate specificity of Grc3, an inexpensive assay that allowed testing of different oligonucleotide substrates was required. Therefore, a PNK phosphorylation assay using fluorescently-labeled substrates was developed. This assay was successfully used to determine that Grc3 can utilize any NTP for phosphoryl transfer activity, but favors ATP24. This protocol adapts the original assay to measure PNK activity of Grc3 on an RNA mimic of its pre-ribosomal RNA substrate (SC-ITS2, Table 1). One challenging aspect of this fluorescence-based approach is the reliance on large polyacrylamide gels to effectively resolve phosphorylated and nonphosphorylated substrates. The protocol provides specific details on how to pour these large gels and avoid common pitfalls when doing so.
Working with RNA requires particular care because it is strongly susceptible to degradation. There are simple preventative steps one can take to limit ribonuclease contamination. A separate RNA workstation that can be easily treated with an RNase inhibitor-containing cleaning agent is often helpful. Always wearing gloves when handling samples and use of RNase-free certified consumables is necessary. Because water is another common source of contamination, it is best to use freshly purified water and sterilize all solutions using a 0.22 &#956;m filter.

<table><tbody><tr><td>0.4 mm 34-well comb</td><td>BioRad</td><td>1653848</td><td></td></tr><tr><td>0.4 mm spacer</td><td>BioRad</td><td>1653812</td><td></td></tr><tr><td>0.5 M EDTA ph 8.0</td><td>KD Medical</td><td>RGF-3130</td><td></td></tr><tr><td>1M Magnesium Chloride</td><td>KD Medical</td><td>CAC-5290</td><td></td></tr><tr><td>1M Tris pH 8.0</td><td>KD Medical</td><td>RGF-3360</td><td></td></tr><tr><td>40% Acrylamide/Bis Solution 29:1</td><td>BioRad</td><td>1610146</td><td></td></tr><tr><td>5M Sodium Chloride</td><td>KD Medical</td><td>RGF-3720</td><td></td></tr><tr><td>ammonium persulfate (APS)</td><td>BioRad</td><td>161-0700</td><td></td></tr><tr><td>ATP</td><td>Sigma</td><td>A2383-1G</td><td></td></tr><tr><td>boric acid</td><td>Sigma</td><td>B0394</td><td></td></tr><tr><td>bromophenol blue sodium salt</td><td>Sigma</td><td>B5525-5G</td><td></td></tr><tr><td>Glass Plates</td><td>Thomas Scientific</td><td>1188K51</td><td></td></tr><tr><td>Hoefer SQ3 Sequencer</td><td>Hoefer</td><td>N/A</td><td></td></tr><tr><td>Image J Software</td><td>N/A</td><td>N/A</td><td>&lt;a href=&quot;https://imagej.nih.gov/ij/&quot; target=&quot;_blank&quot;&gt;https://imagej.nih.gov/ij/&lt;/a&gt;</td></tr><tr><td>Labeled RNA oligonucleotides</td><td>IDT</td><td>Custom Order</td><td></td></tr><tr><td>Pharmacia EPS 3500 Power Supply</td><td>Pharmacia</td><td>N/A</td><td></td></tr><tr><td>Steriflip 0. 22 um Filter</td><td>Millipore</td><td>5FCP00525</td><td></td></tr><tr><td>TEMED</td><td>BioRad</td><td>161-0800</td><td></td></tr><tr><td>tris base</td><td>Sigma</td><td>TRIS-RO</td><td></td></tr><tr><td>Typhoon FLA 9500 gel imager</td><td>GE Healthcare</td><td>N/A</td><td></td></tr><tr><td>Ultra Pure DEPC Water</td><td>Invitrogen</td><td>750023</td><td></td></tr><tr><td>Ultra Pure Glycerol</td><td>Invitrogen</td><td>19E1056865</td><td></td></tr><tr><td>urea</td><td>Fisher Chemical</td><td>U15-500</td><td></td></tr></tbody></table>

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.

A successful representative denaturing gel of a titration of ATP with a fixed amount of Las1-Grc3 complex is shown in Figure 1. Addition of enzyme resulted in Las1-mediated RNA cleavage of the SC-ITS2 RNA substrate, leading to a defined RNA fragment (5-OH C2 RNA). Upon the addition of ATP, the C2 RNA fragment was phosphorylated by Grc3 PNK (5-P C2 RNA). In denaturing gels the phosphorylated RNA migrates faster than its unphosphorylated counterpart. As shown i...

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Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

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 ...

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Research

JoVE Journal

Biochemistry

4.6K Views.  National Institutes of Health. Deze methode kan antwoord geven op belangrijke vragen over de fosforylatie van het vijfste hoofdeinde van DNA- of RNA-moleculen door een speciale klasse van enzymen die bekend staan als polynucleotide kinase. Het belangrijkste voordeel van deze techniek is dat het de resolutie heeft om zeer kleine veranderingen in DNA en RNA substraten te detecteren. Begin met het voorbereiden van de RNA enzym kinase reacties.Combineer voor elke reactie één microliter van 500 nanomolar RNA-substraat....

Video: Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

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.

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 ...

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 ...

Oligopeptide Competition Assay for Phosphorylation Site Determination

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects biological functions and characteristics of the cell. Currently, the most common method for discovering phosphorylation sites is by liquid chromatography/mass spectrometry (LC/MS) analysis, a rapid and sensitive method. However, relatively labile phosphate moieties are often released from phosphopeptides during the fragmentation step, which often yields false-negative signals. In such cases, a traditional in vitro kinase assay using site-directed mutants would be more accurate, but this method is laborious and time-consuming. Therefore, an alternative method using peptide competition may be advantageous. The consensus recognition motif of 5&#39; adenosine monophosphate-activated protein kinase (AMPK) has been established1 and was validated using a positional scanning peptide library assay2. Thus, AMPK phosphorylation sites for a novel substrate could be predicted and confirmed by the peptide competition assays. In this report, we describe the detailed steps and procedures for the in vitro oligopeptide-competing kinase assay by illustrating AMPK-mediated nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation. To authenticate the phosphorylation site, we carried out a sequential in vitro kinase assay using a site-specific mutant. Overall, the peptide competition assay provides a method to screen multiple potential phosphorylation sites and to identify sites for validation by the phosphorylation site mutants.

Peptide competition assays are widely used in a variety of molecular and immunological experiments. This paper describes a detailed method for an in vitro oligopeptide-competing kinase assay and the associated validation procedures, which may be useful to find specific phosphorylation sites.

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects ...

HPLC-based Assay to Monitor Extracellular Nucleotide/Nucleoside Metabolism in Human Chronic Lymphocytic Leukemia Cells

This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (RP-HPLC) assay developed and validated for the quantification of extracellular purine nucleotides and nucleosides produced by purified chronic lymphocytic leukemia (CLL) cells under different culture conditions. The chromatographic separation of adenosine 5&#39;-monophosphate (AMP), adenosine (ADO) and inosine (INO) is performed at RT on a silica-based, reversed-phase column that is used for polar compound retention. The method includes a binary mobile phase, which consists of 7 mM ammonium acetate and acetonitrile with a flow rate of 1.00 ml/min. The eluates are monitored using a Photodiode Array UV detector set at 260 nm. A standard calibration curve is generated to calculate the equation for the analytical quantification of each purine compound. System control, data acquisition and analysis are then performed. Applying this protocol, AMP, INO and ADO elute at 7, 11 and 11.9 min, respectively, and the total run time for each sample is 20 min. This protocol may be applied to different cell types and cell lines (both suspension and adherent), using culture media as matrix. The advantages are easy and fast sample preparation and the requirement of a small amount of supernatant for analysis. Furthermore, the use of a serum-free medium allows skipping the protein precipitation step with acetonitrile that impacts the final concentration of purine compounds. One of the limitations of the method is the requirement of the equilibration column run before each single sample run, making the total run time of the experiment longer and preventing high throughput screening applications.

The protocol described here represents an easy and reproducible method that employs reverse phase high-performance liquid chromatography (RP-HPLC) to measure purine metabolism on chronic lymphocytic leukemia (CLL) cells cultured under different conditions.

This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (RP-HPLC) assay developed and ...

Chemistry

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

Non-Radioactive In Vitro Cardiac Myosin Light Chain Kinase Assays

Cardiac-specific myosin regulatory light chain kinase (cMLCK) regulates cardiac sarcomere structure and contractility by phosphorylating the ventricular isoform of the myosin regulatory light chain (MLC2v). MLC2v phosphorylation levels are significantly reduced in failing hearts, indicating the clinical importance of assessing the activity of cMLCK and the phosphorylation level of MLC2v to elucidate the pathogenesis of heart failure. This paper describes nonradioactive methods to assess both the activity of cMLCK and MLC2v phosphorylation levels. In vitro kinase reactions are performed using recombinant cMLCK with recombinant calmodulin and MLC2v in the presence of ATP and calcium at 25 &#176;C, which are followed by either a bioluminescent ADP detection assay or a phosphate-affinity SDS-PAGE. In the representative study, the bioluminescent ADP detection assay showed a strict linear increase of the signal at cMLCK concentrations between 1.25 nM to 25 nM. Phosphate-affinity SDS-PAGE also showed a linear increase of phosphorylated MLC2v in the same cMLCK concentration range. Next, the time-dependency of the reactions was examined at the concentration of 5 nM cMLCK. A bioluminescent ADP detection assay showed a linear increase in the signal during 90 min of the reaction. Similarly, phosphate-affinity SDS-PAGE showed a time-dependent increase of phosphorylated MLC2v. The biochemical parameters of cMLCK for MLC2v were determined by a Michaelis-Menten plot using the bioluminescent ADP detection assay. The Vmax was 1.65 &#177; 0.10 mol/min/mol kinase and the average Km was around 0.5 USA &#181;M at 25 &#176;C. Next, the activity of wild type and the dilated cardiomyopathy-associated p.Pro639Valfs*15 mutant cMLCK were measured. The bioluminescent ADP detection assay and phosphate-affinity SDS-PAGE correctly detected defects in cMLCK activity and MLC2v phosphorylation, respectively. In conclusion, a combination of the bioluminescent ADP detection assay and the phosphate-affinity SDS-PAGE is a simple, accurate, safe, low-cost, and flexible method to measure cMLCK activity and the phosphorylation level of MLC2v.

This study describes protocols for nonradiometric methods, a bioluminescent ADP detection assay and a phosphate-affinity SDS-PAGE, to determine the kinase activity of cardiac myosin light chain kinase (cMLCK) and the phosphorylation level of its substrate, myosin regulatory light chain (MLC2v).

Cardiac-specific myosin regulatory light chain kinase (cMLCK) regulates cardiac sarcomere structure and contractility by phosphorylating the ventricular ...

Assays for DNA Enzymes Using Synthetic Oligonucleotides

Using Modified Synthetic Oligonucleotides to Assay Nucleic Acid-Metabolizing Enzymes

The availability of a range of modified synthetic oligonucleotides from commercial vendors has allowed the development of sophisticated assays to characterize diverse properties of nucleic acid metabolizing enzymes that can be run in any standard molecular biology lab. The use of fluorescent labels has made these methods accessible to researchers with standard PAGE electrophoresis equipment and a fluorescent-enabled imager, without using radioactive materials or requiring a lab designed for the storage and preparation of radioactive materials, i.e., a Hot Lab. The optional addition of standard modifications such as phosphorylation can simplify assay setup, while the specific incorporation of modified nucleotides that mimic DNA damages or intermediates can be used to probe specific aspects of enzyme behavior. Here, the design and execution of assays to interrogate several aspects of DNA processing by enzymes using commercially available synthetic oligonucleotides are demonstrated. These include the ability of ligases to join or nucleases to degrade different DNA and RNA hybrid structures, differential cofactor usage by the DNA ligase, and evaluation of the DNA-binding capacity of enzymes. Factors to consider when designing synthetic nucleotide substrates are discussed, and a basic set of oligonucleotides that can be used for a range of nucleic acid ligase, polymerase, and nuclease enzyme assays are provided.

Author Spotlight: Characterizing Novel Enzymes from Extremophiles and Common Pathogens to Understand DNA Repair and Replication

Here, a protocol for assaying nucleic acid metabolizing enzymes is presented, using examples of ligase, nuclease, and polymerase enzymes. The assay utilizes fluorescently labeled and unlabeled oligonucleotides that can be combined to form duplexes mimicking RNA and/or DNA damages or pathway intermediates, allowing for the characterization of enzyme behavior.

The availability of a range of modified synthetic oligonucleotides from commercial vendors has allowed the development of sophisticated assays to ...

Assaying for Inorganic Polyphosphate in Bacteria

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many bacteria. There are a variety of methods for quantifying polyP in biological materials, many of which are either labor-intensive or insensitive, limiting their usefulness. We present here a streamlined method for polyP quantification in bacteria, using a silica membrane column extraction optimized for rapid processing of multiple samples, digestion of polyP with the polyP-specific exopolyphosphatase ScPPX, and detection of the resulting free phosphate with a sensitive ascorbic acid-based colorimetric assay. This procedure is straightforward, inexpensive, and allows reliable polyP quantification in diverse bacterial species. We present representative polyP quantification from the Gram-negative bacterium (Escherichia coli), the Gram-positive lactic acid bacterium (Lactobacillus reuteri), and the mycobacterial species (Mycobacterium smegmatis). We also include a simple protocol for nickel affinity purification of mg quantities of ScPPX, which is not currently commercially available.

We describe a simple method for rapid quantification of inorganic polyphosphate in diverse bacteria, including Gram-negative, Gram-positive, and mycobacterial species.

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many ...

Immunology and Infection

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling1,2, while LRRK2 is implicated in the pathogenesis of Parkinson's disease3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with 32P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32P-orthophosphate. The 32P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (32P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain proteins which encode both GTPase and kinase domains and which are phosphorylated in cells. Here, we present a protocol to label LRRK1 and LRRK2 in cells with 32P orthophosphate, thereby providing a means to measure their overall cellular phophorylation levels.

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, ...

Biology

Denaturing Urea Polyacrylamide Gel Electrophoresis for RNA Analysis: A Technique to Separate Fluorescently Labeled Phosphorylated RNA Oligonucleotides from their Non-Phosphorylated Equivalents

Source: Pillon, M. C., et al., <a href="https://www.jove.com/v/61258">Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates</a>. J. Vis. Exp. (2020)
In this video, we demonstrate a denaturing polyacrylamide gel electrophoresis for the separation of phosphorylated RNA species from their non-phosphorylated counterparts. The RNA species are fluorescently labeled for laser detection on the gel.

Source: Pillon, M. C., et al., Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates. J. Vis. Exp. (2020)
In this ...

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

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