The authors thank all current and former members of the Cobb laboratory for valuable work and discussions, and Dionne Ware for administrative assistance. These studies were supported by National Institutes of Health Grant R37 DK34128 and Welch Foundation Grant I1243 to M.H.C.

1. Kinase Purification Resources and General Immunoprecipitation Pipeline
NOTE: Kinases for use with this assay may be sourced from immunoprecipitates of cultured cells or by recombinant means such as affinity-tagged purification6. Below is a general protocol for flag-tagged immunoprecipitation that may have to be modified depending on the kinase of interest. Everything should be kept on ice when possible.
<ol>
	<li>Add 2 &#181;L of 1 mg/mL antibody to 200 &#181;L of cell lysates (usually ~1 mg/mL total protein concentration) and incubate at 4 &#176;C for 1 h while rocking.</li>
	<li>Wash protein A sepharose beads 2-3 times with lysis buffer (see below) by briefly spinning at 4 &#176;C for 30 s to 1 min at 5,000 x g (&#34;touch spin&#34;), removing supernatant with a pipette, and resuspending beads in buffer.
	<ol>
		<li>Prepare lysis buffer: 50 mM HEPES pH 7.7, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, 0.2 mM sodium orthovanadate, 100 mM sodium fluoride, 50 mM &#946;-glycerophosphate, 0.1% Triton X 100 or 0.1% NP-40.</li>
	</ol>
	</li>
	<li>Add 30 &#181;L of 50% slurry of protein A sepharose beads in lysis buffer to lysates; incubate at 4 &#176;C for 1 h while rocking.</li>
	<li>Touch spin at 4 &#176;C to pellet the beads and remove the supernatant.</li>
	<li>Wash 3 times with 1 mL of bead wash buffer (1 M NaCl, 20 mM Tris pH 7.4).</li>
	<li>Wash once with 1x kinase reaction buffer (10 mM HEPES pH 8.0, 10 mM MgCl2). Remove as much buffer as possible without removing beads.</li>
</ol>
2. Initializing Kinase Reactions
NOTE: For IP kinase assays, ~15 &#181;L of unsuspended beads is a typical starting sample. For recombinant protein kinase assays, typical starting amounts range from 0.1-1.0 &#181;g in 1-10 for 25-50 &#181;L final reaction volumes. Adjust the volumes of recombinant protein samples to be equal with the buffer they are stored in prior to initializing the assay. When using high amounts of kinase, include a carrier protein such as BSA to aid in maintaining protein stability for the duration of the assay.
<ol>
	<li>Prepare the 5x kinase reaction buffer given below: 
	50 mM HEPES pH 8.0 
	50 mM MgCl2 
	50 mM Benzamidine (protease inhibitor) 
	50 mM DTT (reduction agent) 
	250 &#181;M ATP (unlabeled, or &#34;cold&#34;)</li>
	<li>Keep kinase samples on ice in 1.5 mL tubes. Prepare the following reaction mixture in separate, chilled 1.5 mL tubes: 
	21 &#181;L H2O 
	6 &#181;L 5x kinase reaction buffer 
	1 &#181;L radiolabeled (&#34;hot&#34;) ATP 
	2 &#181;L substrate (~5 mg/mL) 
	NOTE: For recombinant kinase assays, volume may be adjusted to accommodate purified protein. Calculate such that final reaction volume is 30 &#181;L. Use this recipe to prepare a master mix. Upon receipt of labeled ATP, dilute stock in H2O to a specific activity of 0.01 mCi/&#181;L prior to adding to reaction mixture.</li>
	<li>To initialize the assay, add entire reaction mixture to kinase sample and incubate reaction at 30 &#176;C for 5 min to 1 h depending on activity of kinase being assayed. 
	NOTE: When working with a kinase whose activity has not been previously assayed, perform a time course of kinase activity with 5 min intervals between time points.</li>
</ol>
3. Reaction Termination and SDS-PAGE
<ol>
	<li>Stop reaction by putting on ice and adding 7.5 &#181;L 5x Laemmli sample buffer 6.</li>
	<li>Heat at 100 &#176;C for 30 s to 2 min in heat block.</li>
	<li>Touch spin and load 20 &#181;L per well on 10-15% SDS-PAGE gel. Run gel long enough to separate kinase and substrate (typically 1 h at constant power of 5 W) Make sure to keep gel apparatus shielded to limit exposure to 32P.
	<ol>
		<li>Here, use homemade gel apparatuses, but standard commercially available mini-gels are perfectly suitable. Use the dimensions as follows: 
		Gels: 8 cm width, 5.5 cm length (resolving), 1.5 mm thickness. 
		Combs: 15 wells, 1.5 mm thickness, 2.9 mm width, 16 mm depth</li>
	</ol>
	</li>
</ol>
4. Gel Staining and Drying
Caution: For all steps be sure to reduce personal exposure to 32P by using radioactive shielding and wearing personal protective equipment. For more information on specific steps some helpful references are included.
<ol>
	<li>Remove the gel from glass/alumina plates and place in 50 mL Coomassie stain (10% glacial acetic acid, 50% methanol, 0.25% R-250 dye) for 1 h on an orbital shaker set to 50 rpm. For this step use a container that is slightly larger than the gel itself.8</li>
	<li>Move the gel from stain to fixing solution (10% glacial acetic acid, 20% methanol) in order to de-stain. Rock the gel in 600-700 mL of fixing solution overnight on orbital shaker at 50 rpm. Place pieces of foam or knotted laboratory wipes in the container with the gel to absorb the Coomassie dye 8,9.</li>
	<li>Remove gel from fixing solution and soak in 200 mL methanol for 1-2 min with gentle agitation until gel turns milky white. This will help prevent cracking during the drying step.</li>
	<li>Wet a 14 cm x 14 cm piece of qualitative filter paper with methanol and place onto slab gel vacuum dryer. Lay the gel down onto the filter paper front side facing up.</li>
	<li>Cover the gel with plastic wrap carefully to avoid wrinkles and air bubbles. Run the dryer for 1.5 h at 80 &#176;C. After the vacuum has been attained open the lid and roll out any air bubbles if needed with a soft rubber brayer.</li>
</ol>
5. Autoradiogram and Scintillation Counts
<ol>
	<li>Remove dried gel and attach an autorad marker, phosphorescent ruler or dots to the filter paper on the side of the gel. If using dots make sure they are in an asymmetric pattern. This will align the gel with the film after exposure and development.</li>
	<li>Use a Geiger counter to check the intensity of the signal. For weaker signals exposure at -70 &#176;C to -80 &#176;C with an intensifying screen will increase the film band density.</li>
	<li>In a dark room put dried gel in a film cassette with film and an intensifying screen in the following order from top to bottom: gel, film, intensifying screen.</li>
	<li>Attach the intensifying screen to the lid of the cassette and tape the gel in place to make this step easier. The intensifying screen emits light when irradiated by the beta particles from the 32P. These light emissions penetrate the film more efficiently than the beta particles.
	<ol>
		<li>Make sure the wavelength emitted from the intensifying screen corresponds to a wavelength the film is most sensitive to.</li>
		<li>Use a Geiger counter to determine how long to leave exposure for: if cpm count is ~100 or below, try overnight at -70 &#176;C. If count is ~10,000, start with a 1 h exposure and optimize from there.</li>
	</ol>
	</li>
	<li>At the conclusion of the exposure remove the film and develop in a medical/X-ray film processor. If the exposure was performed at -70 to -80 &#176;C, either allow the cassette to warm to room temperature to minimize condensation on the film or remove the film immediately before condensation forms10.</li>
	<li>Lay the film over the dried gel/filter paper and align the image of the marker/dots with the markers/dots on the dried gel. Mark on the film the bands corresponding to the protein standards. Label the protein standards and which lanes the reactions are for future reference.</li>
	<li>Excise the bands from the gel that correspond to bands of interest on the film and place them in 7 mL scintillation vials. Add 4 mL of scintillation fluid and count with liquid scintillation counter. Confirm the counter is set to monitor the correct energy spectrum window for 32P. 
	NOTE: Be sure to also excise the band corresponding to substrate from the no-kinase control lane. This will be used to calculate background radiation that will be subtracted from all sample scintillation counts.</li>
</ol>
6. Analysis of Results
NOTE: The autoradiogram provides a qualitative visualization of results. For accurate quantitation, 32P incorporation can be measured with a scintillation counter. Data are usually expressed in terms of relative activity, as shown in Figure 1B. As long as uniform conditions are maintained for all samples, relative measurements of specific activity are sufficient to compare treatments.
<ol>
	<li>When calculating absolute specific activity values, perform a rigorous optimization of all reaction conditions, including kinase, substrate, and cold ATP concentrations, in order to ensure a linear range of activity over a time course (e.g. 2 x [kinase] = 2 x specific activity). For kinases with high specific activity, perform assays with increased substrate concentration in case kinase activity is limited by amount of substrate.</li>
	<li>Calculate the specific activity of kinase of interest in units of nanomoles phosphate transferred per min per mg kinase.
	<ol>
		<li>Subtract blanks from the cpm in the counted bands.</li>
		<li>Calculate the decay of hot ATP: [(1/2)^(t/t1/2)] x 0.95 where t is the number of days since the reference date (should be provided by vendor), t1/2 is the half-life of 32P, which is 14.3 days, and 0.95 reflects the efficiency of the scintillation counter. Example: if using hot ATP that is 28 days old, the decay value should be 0.245.</li>
		<li>Calculate the picomoles (pmol) of total ATP in the assay (usually this is equal to the amount of cold ATP in the reaction): assay volume (&#181;L) x [cold ATP] (&#181;M) = total ATP (pmols). Example: 30 &#181;L x 50 &#181;M = 1500 pmol</li>
		<li>Calculate cpm/pmol ATP: [&#181;L of hot ATP x (2.2 x 107) x decay factor]/ pmol of cold ATP. 
		NOTE: The value of 2.2 x 107 converts 0.01 mCi/&#181;L of ATP to cpm.</li>
		<li>Calculate the amount of kinase in the assay in mg. For both recombinant kinase and immunoprecipitated kinases, standard methods such as BCA assays are suitable to determine starting concentrations. Example: wtERK2 0.0002 &#181;g/&#181;L in a 50 &#181;L kinase reaction is 0.01 &#181;g, or 1 x 10-5 mg.</li>
		<li>Calculate total cpm in assay. Assemble 30 &#181;L reactions and run 20 &#181;L from each reaction on a gel. Multiply the cpm counted (after blank subtraction) by a factor of total assay volume/ volume loaded. Continuing the above example, multiply all counts by 1.5, or 30/20.</li>
		<li>Calculate the specific activity of the assayed kinase: 
		Total cpm in assay)/(cpm/pmol ATP)/(assay time in minutes)/(amount of kinase in mg) 
		NOTE: Dividing the above value by 1,000 yields the specific activity in nanomoles/minute/mg</li>
	</ol>
	</li>
	<li>Calculate how many mols of phosphate are incorporated into a substrate (as long as its molecular weight is known). This is used to determine the number of phosphosites on a particular protein once the reaction has gone to completion. 
	Total cpm in assay)/(cpm/pmol ATP)]/(amount of substrate in pmols)</li>
</ol>

<ol>
	<li>Raman, M., Chen, W., Cobb, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+and+properties+of+MAPKs.">Differential regulation and properties of MAPKs.</a> Oncogene. 26 (22), 3100-3112 (2007).</li><li>Wellbrock, C., Arozarena, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Complexity+of+the+ERK/MAP-Kinase+Pathway+and+the+Treatment+of+Melanoma+Skin+Cancer.">The Complexity of the ERK/MAP-Kinase Pathway and the Treatment of Melanoma Skin Cancer.</a> Front Cell Dev Biol. 4, 33 (2016).</li><li>Yang, T., Terman, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+PKA-Mediated+Phosphorylation+of+Plexin+Using+Purified+Proteins.">Characterizing PKA-Mediated Phosphorylation of Plexin Using Purified Proteins.</a> Methods Mol Biol. 1493, 147-159 (2017).</li><li>Ross, H., Armstrong, C. G., Cohen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+non-radioactive+method+for+the+assay+of+many+serine/threonine-specific+protein+kinases.">A non-radioactive method for the assay of many serine/threonine-specific protein kinases.</a> Biochem J. 366 (Pt 3), 977-981 (2002).</li><li>Akita, S., Umezawa, N., Kato, N., Higuchi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Array-based+fluorescence+assay+for+serine/threonine+kinases+using+specific+chemical+reaction.">Array-based fluorescence assay for serine/threonine kinases using specific chemical reaction.</a> Bioorg Med Chem. 16 (16), 7788-7794 (2008).</li><li>Dalby, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Phosphorylation:+Selected+Methods+in+Enzymology.">Protein Phosphorylation: Selected Methods in Enzymology.</a> J Am Chem Soc. 121 (51), 12215 (1999).</li><li>. Autoradiography Available from: <a target="_blank" href="https://www.nationaldiagnostics.com/electrophoresis/article/autoradiography">https://www.nationaldiagnostics.com/electrophoresis/article/autoradiography</a> (2011)</li><li>Walker, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Protein Protocols Handbook. , (2009).</li><li>Xu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=WNK1,+a+novel+mammalian+serine/threonine+protein+kinase+lacking+the+catalytic+lysine+in+subdomain+II.">WNK1, a novel mammalian serine/threonine protein kinase lacking the catalytic lysine in subdomain II.</a> J Biol Chem. 275 (22), 16795-16801 (2000).</li><li>McReynolds, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+or+Mutation+of+the+ERK2+Activation+Loop+Alters+Oligonucleotide+Binding.">Phosphorylation or Mutation of the ERK2 Activation Loop Alters Oligonucleotide Binding.</a> Biochemistry. 55 (12), 1909-1917 (2016).</li><li>Carlson, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+discovery+of+ERK2+substrates+identifies+ERK-mediated+transcriptional+regulation+by+ETV3.">Large-scale discovery of ERK2 substrates identifies ERK-mediated transcriptional regulation by ETV3.</a> Sci Signal. 4 (196), rs11 (2011).</li><li>Courcelles, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphoproteome+dynamics+reveal+novel+ERK1/2+MAP+kinase+substrates+with+broad+spectrum+of+functions.">Phosphoproteome dynamics reveal novel ERK1/2 MAP kinase substrates with broad spectrum of functions.</a> Mol Syst Biol. 9, 669 (2013).</li><li>Myers, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Throughput+Screening+and+Hit+Validation+of+Extracellular-Related+Kinase+5+(ERK5)+Inhibitors.">High-Throughput Screening and Hit Validation of Extracellular-Related Kinase 5 (ERK5) Inhibitors.</a> ACS Comb Sci. 18 (8), 444-455 (2016).</li></ol>

The authors have nothing to disclose.

Kinases are a diverse family of proteins that have evolved extensive functionality in numerous contexts, and kinase assays have been incredibly useful in studying several signaling proteins and have greatly contributed to our current understanding of cellular communication. Notably, the same basic assay was used in characterizing two disparate kinases despite major differences in structure and activity. WNK1 kinase contains an atypical catalytic pocket where the critical lysine has shifted to a unique position and is kno...

Protein kinases are sophisticated enzymes critical for the transmission of cellular signals into appropriate responses1. Given their roles in maintaining homeostasis and the prevention or promotion of disease states2, biochemical methods for assessing kinase activity continue to be powerful tools for delineating the particulars of eukaryotic signaling3. Although strategies utilizing phospho-specific antibodies have been highly informative in terms of measuring the effects of different treatment conditions on cell signaling status4, a kinase assay allows for measuring the effects of different treatment conditions directly in terms of the enzymatic activity of a kinase of interest. While there are several options for similar assays that do not use radioactive materials5, we continue to rely on this method for robust quantitation of results. There are two typical applications of this assay, both valuable for different reasons: the immunoprecipitated (IP) kinase assay (Figure 1) and the recombinant protein kinase assay (Figure 2).
The IP kinase assay is tremendously useful for identifying factors capable of activating specific protein kinases as well as gauging inhibitory treatment conditions. Briefly, an epitope-tagged kinase of interest is transfected into cultured eukaryotic cells, subjected to a variety of treatments, immunoprecipitated, and assayed for the ability to incorporate radiolabeled phosphate into a model substrate (e.g. myelin basic protein (MBP)). IP kinase assay can also be performed without resorting to overexpression, either by immunoprecipitation of endogenous protein or any number of genome-editing techniques. Because treatments are administered in culture, this method can detect stimulations transmitted through multiple upstream factors or parallel pathways by in vitro readout. One major advantage of this method is that it does not require prior knowledge of direct upstream or downstream factors or phosphorylation sites therein. Moreover, once specific substrates for a kinase of interest have been identified, the same kinase assay protocol can be used with recombinant components to measure specific activity towards natural substrates and identify specific phosphorylation sites when combined with mass spectrometry analysis. Sites identified in this manner can be further validated with kinase assays utilizing substrate mutants. Lastly, this method can also be used to detect and measure autophosphorylation.
The protocol provided here assumes either an optimized protein purification scheme or transfection method for expressing an affinity-tagged kinase of interest in cultured cells. For more detailed expositions of transfection, lysis, immunoprecipitation, and protein purification protocols, we suggest referring to Cold Spring Harbor Protocols6. For more information regarding assay development and modification, please refer to Protein Phosphorylation: Selected Methods in Enzymology7.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Protein A Sepharose CL-4B</td>
			<td>GE Healthcare Life Sciences</td>
			<td>17-0963-03</td>
		</tr>
		<tr>
			<td>Radiolabeled [&#947;-32P] ATP</td>
			<td>Perkin Elmer</td>
			<td>NEG035C010MC</td>
		</tr>
		<tr>
			<td>Laemmli Buffer</td>
			<td>Bio-Rad</td>
			<td>#1610737</td>
		</tr>
		<tr>
			<td>Radioactive shielding</td>
			<td>Research Products International</td>
			<td>BR-006</td>
		</tr>
		<tr>
			<td>R-250 dye (Coomassie)</td>
			<td>Thermo Fisher</td>
			<td>20278</td>
		</tr>
		<tr>
			<td>Whatman Grade 3 qualitative filter paper</td>
			<td>Whatman</td>
			<td>1003-917</td>
		</tr>
		<tr>
			<td>Slab gel vacuum dryer</td>
			<td>Bio-Rad</td>
			<td>Model 583 Gel Dryer #1651745&#160;</td>
		</tr>
		<tr>
			<td>Autorad marker</td>
			<td>Agilent Technologies</td>
			<td>420201</td>
		</tr>
		<tr>
			<td>Phosphorescent ruler</td>
			<td>Sigma Aldrich</td>
			<td>R8133</td>
		</tr>
		<tr>
			<td>Phosphorescent dots</td>
			<td>Sigma Aldrich</td>
			<td>L5149</td>
		</tr>
		<tr>
			<td>Geiger counter</td>
			<td>Ludlum</td>
			<td>Model 3 with 44-9 detector</td>
		</tr>
		<tr>
			<td>BioMax Cassette 8x10&#34;</td>
			<td>Carestream Health</td>
			<td>107 2263</td>
		</tr>
		<tr>
			<td>BioMax MS Film 8x10&#34;</td>
			<td>Carestream Health</td>
			<td>829 4985</td>
		</tr>
		<tr>
			<td>BioMax MS Intensifying Screen 8x10&#34;</td>
			<td>Carestream Health</td>
			<td>851 8706</td>
		</tr>
		<tr>
			<td>Medical/X-ray film processor</td>
			<td>Konica Minolta</td>
			<td>SRX-101A</td>
		</tr>
		<tr>
			<td>Scintillation vials</td>
			<td>Research Products International</td>
			<td>125500</td>
		</tr>
		<tr>
			<td>Scintillation fluid</td>
			<td>MP Biomedicals</td>
			<td>188245305</td>
		</tr>
		<tr>
			<td>Beckman LS 6500 Scintillation counter</td>
			<td>Beckman</td>
			<td>LS 6500</td>
		</tr>
	</tbody>
</table>

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.

WNK1 IP kinase assays
Myc-tagged WNK1 was transfected into HEK293 cells and immunoprecipitated with an anti-Myc antibody11. The immunoprecipitate displayed kinase activity towards the model substrate MBP as well as toward itself (Figure 1A). WNK1 mutants were then tested for kinase activity towards MBP by the same method, this time employing GST-tagged constructs (Figure 1B)...

Watch this Scientific Journal Video about Assaying Protein Kinase Activity with Radiolabeled ATP at JoVE.com

Assaying Protein Kinase Activity with Radiolabeled ATP

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 ...

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18.2K Views.  University of Texas Southwestern Medical Center. 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 ([γ-32P] ATP), a reliable method to aid in elucidation of cellular signaling regulation.

Video: Assaying Protein Kinase Activity with Radiolabeled ATP

Investigating Protein Sequence-structure-dynamics Relationships with Bio3D-web

We demonstrate the usage of Bio3D-web for the interactive analysis of biomolecular structure data. The Bio3D-web application provides online functionality for: (1) The identification of related protein structure sets to user specified thresholds of similarity; (2) Their multiple alignment and structure superposition; (3) Sequence and structure conservation analysis; (4) Inter-conformer relationship mapping with principal component analysis, and (5) comparison of predicted internal dynamics via ensemble normal mode analysis. This integrated functionality provides a complete online workflow for investigating sequence-structure-dynamic relationships within protein families and superfamilies.

A protocol for the online investigation of protein sequence-structure-dynamics relationships using Bio3D-web is presented.

We demonstrate the usage of Bio3D-web for the interactive analysis of biomolecular structure data. The Bio3D-web application provides online functionality ...

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; ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson's Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation

The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson&#8217; disease (PD) and all pathogenic LRRK2 mutations result in hyperactivation of its kinase function. Here, we describe an easy and robust assay to quantify LRRK2 kinase pathway activity in human peripheral blood neutrophils by measuring LRRK2-controlled phosphorylation of one of its physiological substrates, Rab10 at threonine 73. The immunoblotting analysis described requires a fully selective and phosphospecific antibody that recognizes the Rab10 Thr73 epitope phosphorylated by LRRK2, such as the MJFF-pRab10 rabbit monoclonal antibody. It uses human peripheral blood neutrophils, because peripheral blood is easily accessible and neutrophils are an abundant and homogenous constituent. Importantly, neutrophils express relatively high levels of both LRRK2 and Rab10. A potential drawback of neutrophils is their high intrinsic serine protease activity, which necessitates the use of very potent protease inhibitors such as the organophosphorus neurotoxin diisopropylfluorophosphate (DIFP) as part of the lysis buffer. Nevertheless, neutrophils are a valuable resource for research into LRRK2 kinase pathway activity in vivo and should be considered for inclusion into PD biorepository collections.

Mutations in the leucine rich repeat kinase 2 gene (LRRK2) cause hereditary Parkinson&#8217;s disease. We have developed an easy and robust method for assessing LRRK2-controlled phosphorylation of Rab10 in human peripheral blood neutrophils. This may help identify individuals with increased LRRK2 kinase pathway activity.

The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson&#8217; disease (PD) and all pathogenic LRRK2 ...

Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets in both physiological and pathological states. CK2 is an evolutionarily conserved serine/threonine kinase with a growing list of hundreds of substrates involved in multiple cellular processes. Due to its pleiotropic properties, identifying and characterizing a comprehensive set of CK2 substrates has been particularly challenging and remains a hurdle in the study of this important enzyme. To address this challenge, we have devised a versatile experimental strategy that enables the targeted enrichment and identification of putative CK2 substrates. This protocol takes advantage of the unique dual co-substrate specificity of CK2 allowing for specific thiophosphorylation of its substrates in a cell or tissue lysate. These substrate proteins are subsequently alkylated, immunoprecipitated, and identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). We have previously used this approach to successfully identify CK2 substrates from Drosophila ovaries and here we extend the application of this protocol to human glioblastoma cells, illustrating the adaptability of this method to investigate the biological roles of this kinase in various model organisms and experimental systems.

The objective of this protocol is to label, enrich, and identify substrates of protein kinase CK2 from a complex biological sample such as a cell lysate or tissue homogenate. This method leverages unique aspects of CK2 biology for this purpose.

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets ...

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral RNAs. When bound to viral double-stranded RNAs (dsRNAs), PKR undergoes dimerization and subsequent autophosphorylation. Phosphorylated PKR (pPKR) becomes active and induces phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2&#945;) to suppress global translation. Increasing evidence suggests that PKR can be activated under physiological conditions such as during the cell cycle or under various stress conditions without infection. However, our understanding of the RNA activators of PKR is limited due to the lack of a standardized experimental method to capture and analyze PKR-interacting dsRNAs. Here, we present an experimental protocol to specifically enrich and analyze PKR bound RNAs during the cell cycle using HeLa cells. We utilize the efficient crosslinking activity of formaldehyde to fix PKR-RNA complexes and isolate them via immunoprecipitation. PKR co-immunoprecipitated RNAs can then be further processed to generate a high-throughput sequencing library. One major class of PKR-interacting cellular dsRNAs is mitochondrial RNAs (mtRNAs), which can exist as intermolecular dsRNAs through complementary interaction between the heavy-strand and the light-strand RNAs. To study the strandedness of these duplex mtRNAs, we also present a protocol for strand-specific qRT-PCR. Our protocol is optimized for the analysis of PKR-bound RNAs, but it can be easily modified to study cellular dsRNAs or RNA-interactors of other dsRNA binding proteins.

We present experimental approaches for studying RNA-interactors of double-stranded RNA binding protein kinase RNA-activated (PKR) during the mammalian cell cycle using HeLa cells. This method utilizes formaldehyde to crosslink RNA-PKR complexes and immunoprecipitation to enrich PKR-bound RNAs. These RNAs can be further analyzed through high-throughput sequencing or qRT-PCR.

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. Y-27632 is a highly potent inhibitor of Rho-associated, coiled-coil-containing protein kinase (RhoA/ROCK) and is used to prevent dissociation-induced cell apoptosis (anoikis). We demonstrate that Y-27632 pretreatment for human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs+RI) prior to implantation results in a cell engraftment rate improvement in a mouse model of acute myocardial infarction (MI). Here, we describe a complete procedure of hiPSC-CMs differentiation, purification, and cell pretreatment with Y-27632, as well as the resulting cell contraction, calcium transient measurements, and transplantation into mouse MI models. The proposed method provides a simple, safe, effective, and low-cost method which significantly increases the cell engraftment rate. This method cannot only be used in conjunction with other methods to further enhance the cell transplantation efficiency but also provides a favorable basis for the study of the mechanisms of other cardiac diseases.

In this protocol, we demonstrate and elaborate on how to use human induced pluripotent stem cells for cardiomyocyte differentiation and purification, and further, on how to improve its transplantation efficiency with Rho-associated protein kinase inhibitor pretreatment in a mouse myocardial infarction model.

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. ...

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 ...

Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the dissection and manipulation of biological pathways. Currently, only a limited number of chemically induced dimerization (CID) systems exist and engineering new ones with desired sensitivity and selectivity for specific small-molecule ligands remains a challenge in the field of protein engineering. We here describe a high throughput screening method, combinatorial binders-enabled selection of CID (COMBINES-CID), for the de novo engineering of CID systems applicable to a large variety of ligands. This method uses the two-step selection of a phage-displayed combinatorial nanobody library to obtain 1) &#34;anchor binders&#34; that first bind to a ligand of interest and then 2) &#34;dimerization binders&#34; that only bind to anchor binder-ligand complexes. To select anchor binders, a combinatorial library of over 109 complementarity-determining region (CDR)-randomized nanobodies is screened with a biotinylated ligand and hits are validated with the unlabeled ligand by bio-layer interferometry (BLI). To obtain dimerization binders, the nanobody library is screened with anchor binder-ligand complexes as targets for positive screening and the unbound anchor binders for negative screening. COMBINES-CID is broadly applicable to select CID binders with other immunoglobulin, non-immunoglobulin, or computationally designed scaffolds to create biosensors for in vitro and in vivo detection of drugs, metabolites, signaling molecules, etc.

Creating chemically induced protein dimerization systems with desired affinity and specificity for any given small molecule ligand would have many biological sensing and actuation applications. Here, we describe an efficient, generalizable method for de novo engineering of chemically induced dimerization systems via the stepwise selection of a phage-displayed combinatorial single-domain antibody library.