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.

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

Watch this Scientific Journal Video about Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates at JoVE.com

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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Biochemistry

4.6K Views.  National Institutes of Health. This protocol describes a nonradioactive assay to measure kinase activity of polynucleotide kinases (PNKs) on small DNA and RNA substrates.

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

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 ...

Quantitative Immunofluorescence to Measure Global Localized Translation

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and organogenesis, as well as in multiple diseases. Numerous publications have convincingly shown that specific mechanisms tightly regulate mRNA translation. Increased interest in the translation-induced regulation of protein expression has led to the development of novel methods to study and follow de novo protein synthesis in cellulo. However, most of these methods are complex, making them costly and often limiting the number of mRNA targets that can be studied. This manuscript proposes a method that requires only basic reagents and a confocal fluorescence imaging system to measure and visualize the changes in mRNA translation that occur in any cell line under various conditions. This method was recently used to show localized translation in the subcellular structures of adherent cells over a short period of time, thus offering the possibility of visualizing de novo translation for a short period during a variety of biological processes or of validating changes in translational activity in response to specific stimuli.

This manuscript describes a method to visualize and quantify localized translation events in subcellular compartments. The approach proposed in this manuscript requires a basic confocal imaging system and reagents and is rapid and cost-effective.

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

Assay for Phosphorylation and Microtubule Binding Along with Localization of Tau Protein in Colorectal Cancer Cells

The microtubule-associated protein tau is a neuronal protein that localizes mostly in axons. Generally tau is essential for normal neuronal functioning because it is involved in microtubule assembly and stabilization. Besides neurons, tau is expressed in human breast, prostate, gastric, colorectal, and pancreatic cancers where it shows nearly similar structure and exerts similar functions as the neuronal tau. The amount of tau and its phosphorylation can change its function as a stabilizer of microtubules, and lead to the development of paired helical filaments in different neurodegenerative disorders, such as Alzheimer&#39;s disease. Determining the phosphorylation state of tau and its microtubule-binding characteristics is important. In addition, examining the intracellular localization of tau is important in different diseases. This manuscript details standard protocols for measuring tau phosphorylation and tau binding to microtubules in colorectal cancer cells with or without curcumin and LiCl treatment. These treatments can be used to stop cancer cell proliferation and development. Intracellular localization of tau is examined by using immunohistochemistry and confocal microscopy while using low amounts of antibodies. These assays can be used repetitively for screening compounds that affect tau hyperphosphorylation or microtubule binding. Novel therapeutics used for different tauopathies or related anticancer agents can potentially be characterized using these protocols.

This manuscript describes standard protocols for measuring tau hyperphosphorylation, measuring tau binding to microtubules, and localization of intracellular tau following drug treatments. These protocols can be used repetitively for screening drugs or other compounds that target tau hyperphosphorylation or microtubule binding.

The microtubule-associated protein tau is a neuronal protein that localizes mostly in axons. Generally tau is essential for normal neuronal functioning ...

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; ...

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have developed a simple non-labeled and non-radio-isotopic method using a matrix-assisted laser desorption ionization with time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis for a proofreading study. Here, a DNA polymerase [e.g., the Klenow fragment (KF) of Escherichia coli DNA polymerase I (pol I) in this study] in the presence of all four dideoxyribonucleotide triphosphates is used to process a mismatched primer-template duplex. The mismatched primer is then proofread/extended and subjected to MALDI-TOF MS. The products are distinguished by the mass change of the primer down to single nucleotide variations. Importantly, a proofreading can also be determined for internal single mismatches, albeit at different efficiencies. Mismatches located at 2-4-nucleotides (nt) from the 3' end were efficiently proofread by pol I, and a mismatch at 5 nt from the primer terminus showed only a partial correction. No proofreading occurred for internal mismatches located at 6 - 9 nt from the primer 3' end. This method can also be applied to DNA repair assays (e.g., assessing a base-lesion repair of substrates for the endo V repair pathway). Primers containing 3' penultimate deoxyinosine (dI) lesions could be corrected by pol I. Indeed, penultimate T-I, G-I, and A-I substrates had their last 2 dI-containing nucleotides excised by pol I before adding a correct ddN 5'-monophosphate (ddNMP) while penultimate C-I mismatches were tolerated by pol I, allowing the primer to be extended without repair, demonstrating the sensitivity and resolution of the MS assay to measure DNA repair.

A non-labeled, non-radio-isotopic method for DNA polymerase proofreading and a DNA repair assay was developed by using high-resolution MALDI-TOF mass spectrometry and a single nucleotide extension strategy. The assay proved to be very specific, simple, rapid, and easy to perform for proofreading and repair patches shorter than 9-nucleotides.

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have ...

Dual DNA Rulers to Study the Mechanism of Ribosome Translocation with Single-Nucleotide Resolution

The ribosome translocation refers to the ribosomal movement on the mRNA by exactly three nucleotides (nt), which is the central step in protein synthesis. To investigate its mechanism, there are two essential technical requirements. First is single-nt resolution that can resolve normal translocation from frameshifting, during which the ribosome moves by other than 3 nt. The second is the capability to probe both the entrance and exit sides of mRNA in order to elucidate the whole picture of translocation. We report the dual DNA ruler assay that is based on the critical dissociation forces of DNA-mRNA duplexes, obtained by force-induced remnant magnetization spectroscopy (FIRMS).&#160; With 2-4 pN force resolution, the dual ruler assay is sufficient to distinguish different translocation steps. By implementing a long linker on the probing DNAs, they can reach the mRNA on the opposite side of the ribosome, so that the mRNA position can be determined for both sides. Therefore, the dual ruler assay is uniquely suited to investigate the ribosome translocation, and nucleic acid motion in general. We show representative results which indicated a looped mRNA conformation and resolved normal translocation from frameshifting.

Dual DNA ruler assay is developed to determine the mRNA position during ribosome translocation, which relies on the dissociation forces of the formed DNA-mRNA duplexes. With single-nucleotide resolution and capability of reaching both ends of mRNA, it can provide mechanistic insights for ribosome translocation and probe other nucleic acid displacements.

The ribosome translocation refers to the ribosomal movement on the mRNA by exactly three nucleotides (nt), which is the central step in protein synthesis. ...

Using an Extracellular Flux Analyzer to Measure Changes in Glycolysis and Oxidative Phosphorylation during Mouse Sperm Capacitation

Mammalian sperm acquire fertilization capacity in the female reproductive tract in a process known as capacitation. Capacitation-associated processes require energy. There remains an ongoing debate about the sources generating the ATP which fuels sperm progressive motility, capacitation, hyperactivation, and acrosome reaction. Here, we describe the application of an extracellular flux analyzer as a tool to analyze changes in energy metabolism during mouse sperm capacitation. Using H+- and O2- sensitive fluorophores, this method allows monitoring glycolysis and oxidative phosphorylation in real-time in non-capacitated versus capacitating sperm. Using this assay in the presence of different energy substrates and/or pharmacological activators and/or inhibitors can provide important insights into the contribution of different metabolic pathways and the intersection between signaling cascades and metabolism during sperm capacitation.

We describe the application of an extracellular flux analyzer to monitor real-time changes in glycolysis and oxidative phosphorylation during mouse sperm capacitation.

Mammalian sperm acquire fertilization capacity in the female reproductive tract in a process known as capacitation. Capacitation-associated processes ...

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. This method combines expression of proteins tagged with the fluorescent non-canonical amino acid ANAP, and FRET between ANAP and fluorescent (trinitrophenyl) nucleotide derivatives. We present examples of nucleotide binding to ANAP-tagged KATP ion channels measured in unroofed plasma membranes and excised, inside-out membrane patches under voltage clamp. The latter allows for simultaneous measurements of ligand binding and channel current, a direct readout of protein function. Data treatment and analysis are discussed extensively, along with potential pitfalls and artefacts. This method provides rich mechanistic insights into the ligand-dependent gating of KATP channels and can readily be adapted to the study of other nucleotide-regulated proteins or any receptor for which a suitable fluorescent ligand can be identified.

This protocol presents a method for measuring adenine nucleotide binding to receptors in real time in a cellular environment. Binding is measured as F&#246;rster resonance energy transfer (FRET) between trinitrophenyl nucleotide derivatives and protein labeled with a non-canonical, fluorescent amino acid.

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. ...

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this enzyme can hydrolyze dNTPs into their corresponding nucleosides and inorganic triphosphates. Due to its critical role in nucleotide metabolism, its association to several pathologies, and its role in therapy resistance, intense research is currently being carried out for a better understanding of both the regulation and cellular function of this enzyme. For this reason, development of simple and inexpensive high-throughput amenable methods to probe small molecule interaction with SAMHD1, such as allosteric regulators, substrates, or inhibitors, is vital. To this purpose, the enzyme-coupled malachite green assay is a simple and robust colorimetric assay that can be deployed in a 384-microwell plate format allowing the indirect measurement of SAMHD1 activity. As SAMHD1 releases the triphosphate group from nucleotide substrates, we can couple a pyrophosphatase activity to this reaction, thereby producing inorganic phosphate, which can be quantified by the malachite green reagent through the formation of a phosphomolybdate malachite green complex. Here, we show the application of this methodology to characterize known inhibitors of SAMHD1 and to decipher the mechanisms involved in SAMHD1 catalysis of non-canonical substrates and regulation by allosteric activators, exemplified by nucleoside-based anticancer drugs. Thus, the enzyme-coupled malachite green assay is a powerful tool to study SAMHD1, and furthermore, could also be utilized in the study of several enzymes which release phosphate species.

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase with critical roles in human health and disease. Here we present a versatile enzyme-coupled SAMHD1 activity assay, deployed in a 384-well microplate format, that allows for the evaluation of small molecules and nucleotide analogues as SAMHD1 substrates, activators, and inhibitors.

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this ...

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 ...