Name: quantification of bacterial histidine kinase autophosphorylation using a nitrocellulose binding assay
Uploaded: 2017-01-11T14:00:00
Description: Watch this Scientific Journal Video about Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay at JoVE.com

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

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

<ol>
	<li>Stock, A. M., Robinson, V. L., Goudreau, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-component+signal+transduction.">Two-component signal transduction.</a> Annu. Rev. Biochem. 69, 183-215 (2000).</li><li>Jung, K., Fried, L., Behr, S., Heermann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histidine+kinases+and+response+regulators+in+networks.">Histidine kinases and response regulators in networks.</a> Curr. Opin. Microbiol. 15 (2), 118-124 (2012).</li><li>Parkinson, J. S., Kofoid, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Communication+modules+in+bacterial+signaling+proteins.">Communication modules in bacterial signaling proteins.</a> Annu. Rev. Genet. 26, 71-112 (1992).</li><li>Laub, M. T., Biondi, E. G., Skerker, 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=Phosphotransfer+profiling:+systematic+mapping+of+two-component+signal+transduction+pathways+and+phosphorelays.">Phosphotransfer profiling: systematic mapping of two-component signal transduction pathways and phosphorelays.</a> Methods Enzymol. 423, 531-548 (2007).</li><li>Ronson, C. W., Nixon, B. T., Ausubel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+domains+in+bacterial+regulatory+proteins+that+respond+to+environmental+stimuli.">Conserved domains in bacterial regulatory proteins that respond to environmental stimuli.</a> Cell. 49 (5), 579-581 (1987).</li><li>Szurmant, H., Bu, L., Brooks, C. L., Hoch, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+essential+sensor+histidine+kinase+controlled+by+transmembrane+helix+interactions+with+its+auxiliary+proteins.">An essential sensor histidine kinase controlled by transmembrane helix interactions with its auxiliary proteins.</a> Proc. Natl. Acad. Sci. USA. 105 (15), 5891-5896 (2008).</li><li>Price, M. S., Chao, L. Y., Marletta, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shewanella+oneidensis+MR-1+H-NOX+regulation+of+a+histidine+kinase+by+nitric+oxide.">Shewanella oneidensis MR-1 H-NOX regulation of a histidine kinase by nitric oxide.</a> Biochemistry. 46 (48), 13677-13683 (2007).</li><li>Zhulin, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+superfamily+of+chemotaxis+transducers:+from+physiology+to+genomics+and+back.">The superfamily of chemotaxis transducers: from physiology to genomics and back.</a> Adv. Microb. Physiol. 45, 157-198 (2001).</li><li>Robinson, V. L., Buckler, D. R., Stock, A. 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+tale+of+two+components:+a+novel+kinase+and+a+regulatory+switch.">A tale of two components: a novel kinase and a regulatory switch.</a> Nature Struct. Biol. 7 (8), 626-633 (2000).</li><li>Attwood, P. V., Piggott, M. J., Zu, X. L., Besant, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focus+on+phosphohistidine.">Focus on phosphohistidine.</a> Amino acids. 32 (1), 145-156 (2007).</li><li>Kee, J. -. M., Muir, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chasing+phosphohistidine,+an+elusive+sibling+in+the+phosphoamino+acid+family.">Chasing phosphohistidine, an elusive sibling in the phosphoamino acid family.</a> ACS Chem. Biol. 7 (1), 44-51 (2012).</li><li>Tawa, P., Stewart, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+CheA+Autophosphorylation+and+Dephosphorylation+Reactions.">Kinetics of CheA Autophosphorylation and Dephosphorylation Reactions.</a> Biochemistry. 33 (25), 7917-7924 (1994).</li><li>Marina, A., Mott, C., Auyzenberg, A., Hendrickson, W. A., Waldburger, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+mutational+analysis+of+the+PhoQ+histidine+kinase+catalytic+domain.+Insight+into+the+reaction+mechanism.">Structural and mutational analysis of the PhoQ histidine kinase catalytic domain. Insight into the reaction mechanism.</a> J. Biol. Chem. 276 (44), 41182-41190 (2001).</li><li>Fuhs, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+1-+and+3-Phosphohistidine+Antibodies:+New+Tools+to+Study+Histidine+Phosphorylation.">Monoclonal 1- and 3-Phosphohistidine Antibodies: New Tools to Study Histidine Phosphorylation.</a> Cell. 162 (1), 198-210 (2015).</li><li>Ueno, T. B., Johnson, R. A., Boon, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+assay+for+the+quantification+of+histidine+kinase+autophosphorylation.">Optimized assay for the quantification of histidine kinase autophosphorylation.</a> Biochem. Biophys. Res. Commun. 465 (3), 331-337 (2015).</li><li>Bradford, 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+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal. Biochem. 7 (72), 248-254 (1976).</li><li>Stoscheck, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+protein.">Quantitation of protein.</a> Methods Enzymol. 182, 50-68 (1990).</li><li>Funt, B. L., Hetherington, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scintillation+counting+of+beta+activity+on+filter+paper.">Scintillation counting of beta activity on filter paper.</a> Science. 131 (3413), 1608-1609 (1960).</li><li>Bem, E. M., Bem, H., Reimch&#252;ssel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+phosphorus-32+and+calcium-45+in+biological+samples+by+&#268;erenkov+and+liquid+scintillation+counting.">Determination of phosphorus-32 and calcium-45 in biological samples by &#268;erenkov and liquid scintillation counting.</a> Int. J. Appl. Radiat. Isot. 31 (6), 371-374 (1980).</li></ol>

The nitrocellulose binding assay we have described has many advantages over previously used methods to characterize histidine kinases. In comparison to traditional SDS/PAGE-based autoradiography, our method is higher throughput and less time-consuming. The nitrocellulose membrane is easier to handle than SDS gels, and does not need to be fixed. Ponceau staining the nitrocellulose allows for the protein spots to be visualized. This provides an easy way to cut out each spot for scintillation counting, and determine that pr...

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

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

quantification of bacterial histidine kinase autophosphorylation using a nitrocellulose binding assay

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

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

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

Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay

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

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

The overall goal of this protocol is to quantify autophosphorylation of purified bacterial histidine kinases. This method can help answer key questions in the field of two-component signaling. Such as the effect of cognate stimuli in protein-protein interactions on autophosphorylation of histidine kinases.The main advantages of this technique are that it's a high throughput method that can be used to accurately detect picomoles of phosphohistadine formed and obtain kinetic parameters for histidine kinases. As a post-doctoral fellow, I use filtration on nitrocellulose membranes to quantify protein-bound radioactive ligands. Consequently, it made sense to develop a similar technique for quantifying histidine phosphorylation in bacterial histidine kinases.Begin this procedure with preparation of reagents and materials as described in the text protocol. To set up the 96 well dot-plot apparatus, cut an eight centimeter by 12 centimeter piece of nitrocellulose membrane. Position the nitrocellulose in the dot-plot apparatus.Ensure that the membrane fits such that the apparatus is sealed and all wells are completely covered by the membrane. To collect the filtrate, connect the apparatus to a secondary sidearm flask. From the valve stem, connect adequate tubing to allow the apparatus to be comfortably used without tipping the secondary flask.Then, connect the secondary sidearm flask to an aspirator vacuum source. Test that the apparatus is completely sealed by applying a vacuum to the apparatus. If the apparatus is correctly assembled, a strong vacuum will be present in all of the wells.All tubing connections can be wrapped in Parafilm or Saran wrap to ensure a tight seal. Prior to initiation, prepare all four reaction components as four X stock solutions, as described in the text protocol. Mix equal volumes of reaction buffer, histidine kinase, and double distilled water.Allow these reaction components to equilibrate at room temperature for a short time. To initiate the reaction, add one volume of gamma-32 P ATP solution and mix by pippetting up and down. Track the elapsed reaction time with a timer and allow the reaction to proceed for the desired time.Quench the reaction. Add an aliquot of the reaction to 325 millimolar phosphoric acid. Immediately place the quenched reactions on ice until all reactions have been terminated.Place the preassembled 96 well dot-plot apparatus into a secondary container. This container should be large enough for the apparatus to be easily disassembled in, as the apparatus and membrane will be radioactive after use. Apply a vacuum to the 96 well dot-plot apparatus.Once the apparatus is under vacuum, carefully pipette the quenched reaction directly onto the nitrocellulose membrane in each well. Repeat this process until all reactions are loaded onto the membrane. Since the binding capacity of nitrocellulose is greater than the amount of spotted histidine kinase, it can be assumed that nearly all of the kinase binds to the membrane when loaded correctly.Depending on the apparatus used, take care not to puncture the nitrocellulose. Observe that all of the reaction has made contact with the nitrocellulose. It is easy for some of the reaction to stick to the walls of the well.Wash the wells with 100 microliters of ice cold 25 millimolar phosphoric acid. This will allow any reaction volume that may have been trapped in the well to reach the membrane, ensuring complete loading of all reactions. Allow the entire volume to flow through the membrane.Carefully disassemble the apparatus before shutting off the vacuum. Be advised that both the apparatus and nitrocellulose membrane are radioactive at this time. Do not remove any apparatus components from the secondary container.With forceps, carefully transfer the nitrocellulose membrane from the apparatus to a container of approximately 200 milliliters of 25 millimolar ice cold phosphoric acid. Place a lid on this container, as the wash will be radioactive. At this time, the vacuum may be shut off.Place the container with the washing membrane on a rocker. Allow the membrane to gently rock for 20 minutes. After 20 minutes, carefully decant the used wash solution into a large bucket to be used for temporary waste storage.Add 200 milliliters of ice cold 25 millimolar phosphoric acid to the membrane. Wash the membrane in this manner at least three times to remove background gamma-32 P ATP signal from the membrane. After the third wash, test the used wash solution for radiation with a Geiger counter.Continue to wash the membrane until no signal is present in the wash solution. After the membrane is sufficiently washed, allow the membrane to briefly air dry, which typically takes five minutes or less. After exposure of the nitrocellulose to a phosphorus green, as described in the text protocol, prepare for Ponceau S staining and destaining.Briefly immerse the nitrocellulose membrane in Ponceau S staining solution for one to two minutes. Wet the entire membrane with the stain prior to destaining. Then, decant the excess Ponceau S from the membrane.Rinse the membrane with double distilled water until only the spots from the histidine kinase are stained. Note that this procedure will only work if all spots contain detectable amounts of protein. Using scissors, cut out each spot on the nitrocellulose membrane.It is not necessary to cut perfectly along the edge of the stained spot. If the membrane was washed sufficiently, background due to varying size of cutout spots is negligible. It is only important to cut out the entire spot for each reaction.Using forceps, carefully transfer each spot into scintillation vials. No scintillation cocktail is necessary, as the phosphorus 32 is readily detectable by Cherenkov radiation. Next, spot several dilutions of gamma-32 P ATP solution onto one centimeter by one centimeter squares of nitrocellulose.Using forceps, carefully transfer each square into scintillation vials sans scintillation cocktail. These samples will be used to generate a standard curve to determine the counts per minute per picomole of the ATP solution. Ponceau staining of the nitrocellulose membrane is shown here.Staining is uniform throughout all samples, indicating that all samples are equally loaded. Fluorography results of the nitrocellulose membrane show the relative level of radiolabeled phosphohistidine in each spot. The fourth and eighth row are negative controls demonstrating that excess ATP is washed from the nitrocellulose membrane.Representative results of quantification of histidine kinase autophosphorylation are shown here. This data is generated from scintillation counting of each spot cut out from the membrane. The standard curve of counts per minute per picomole of ATP shown here is also generated from scintillation counting and is used to calculate the picomoles of phosphohistidine present in each sample.Following this procedure, other methods, like introducing cognate stimuli or proteins that are hypothesized to modulate kinase activity, can be added into the experiment to answer additional questions like the effect of these additives on kinase activity. Don't forget that working with radioactivity can be extremely hazardous. Precautions such as proper radiation safety training and the use of personal protective equipment and shielding should always be made while performing this procedure.

quantification-bacterial-histidine-kinase-autophosphorylation-using

Research

JoVE Journal

Biochemistry

Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of the kinase activity of LRRK2 has been implicated in the pathogenesis of PD, it is essential to establish a method to evaluate the physiological levels of the kinase activity of LRRK2. Recent studies revealed that LRRK2 phosphorylates members of the Rab GTPase family, including Rab10, under physiological conditions. Although the phosphorylation of endogenous Rab10 by LRRK2 in cultured cells could be detected by mass spectrometry, it has been difficult to detect it by immunoblotting due to the poor sensitivity of currently available phosphorylation-specific antibodies for Rab10. Here, we describe a simple method of detecting the endogenous levels of Rab10 phosphorylation by LRRK2 based on immunoblotting utilizing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) combined with a phosphate-binding tag (P-tag), which is N-(5-(2-aminoethylcarbamoyl)pyridin-2-ylmetyl)-N,N&#39;,N&#39;-tris(pyridin-2-yl-methyl)-1,3-diaminopropan-2-ol. The present protocol not only provides an example of the methodology utilizing the P-tag but also enables the assessment of how mutations as well as inhibitor treatment/administration or any other factors alter the downstream signaling of LRRK2 in cells and tissues.

The present study describes a simple method of detecting endogenous levels of Rab10 phosphorylation by leucine-rich repeat kinase 2.

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of ...

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of diverse cellular functions, including cytoskeletal dynamics, cell motility, cell polarity, axonal guidance, vesicular trafficking, and cell cycle control. Changes in Rho GTPase signaling play an essential regulatory role in many pathological conditions, such as&#160;cancer, central nervous system diseases, and immune system-dependent diseases. The posttranslational modification of Rho GTPases (i.e., prenylation by mevalonate pathway intermediates) and GTP binding are key factors which affect the activation of this protein. In this paper, two essential and simple methods are provided to detect a broad range of Rho GTPase prenylation and GTP binding activities. Details of the technical procedures that have been used are explained step by step in this manuscript.

Here we describe a protocol to investigate the prenylation and guanosine-5'-triphosphate (GTP)-loading of Rho GTPase. This protocol consists of two detailed methods, namely membrane fractionation and a GTPase-linked immunosorbent assay. The protocol can be used for measuring the prenylation and GTP loading of different other small GTPases.

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of ...

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; ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

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 ...

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 ...

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 ...

Concanavalin A-Based Sedimentation Assay to Measure Substrate Binding of Glucan Phosphatases

Glucan phosphatases belong to the larger family of dual specificity phosphatases (DSP) that dephosphorylate glucan substrates, such as glycogen in animals and starch in plants. The crystal structures of glucan phosphatase with model glucan substrates reveal distinct glucan-binding interfaces made of DSP and carbohydrate-binding domains. However, quantitative measurements of glucan-glucan phosphatase interactions with physiologically relevant substrates are fundamental to the biological understanding of the glucan phosphatase family of enzymes and the regulation of energy metabolism. This manuscript reports a Concanavalin A (ConA)-based in vitro sedimentation assay designed to detect the substrate binding affinity of glucan phosphatases against different glucan substrates.&#160;As a proof of concept, the dissociation constant (KD) of glucan phosphatase Arabidopsis thaliana Starch Excess4 (SEX4) and amylopectin was determined. The characterization of SEX4 mutants and other members of the glucan phosphatase family of enzymes further demonstrates the utility of this assay to assess the differential binding of protein- carbohydrate interactions. These data demonstrate the suitability of this assay to characterize a wide range of starch and glycogen interacting proteins.

This method describes a lectin-based in vitro sedimentation assay to quantify the binding affinity of glucan phosphatase and amylopectin. This co-sedimentation assay is reliable for measuring glucan phosphatase substrate binding and can be applied to various solubilized glucan substrates.

Glucan phosphatases belong to the larger family of dual specificity phosphatases (DSP) that dephosphorylate glucan substrates, such as glycogen in animals ...

Detection and Quantification of Calcitonin Gene-Related Peptide (CGRP) in Human Plasma Using a Modified Enzyme-Linked Immunosorbent Assay

Calcitonin gene-related peptide (CGRP) is a vasoactive neuropeptide that plays a putative role in the pathophysiology of migraine headaches and may be a candidate for biomarker status. CGRP is released from neuronal fibers upon activation and induces sterile neurogenic inflammation and arterial vasodilation in the vasculature that receives trigeminal efferent innervation. The presence of CGRP in the peripheral vasculature has spurred investigations to detect and quantify this neuropeptide in human plasma using proteomic assays, such as the enzyme-linked immunosorbent assay (ELISA). However, its half-life of 6.9 min and the variability in technical details of assay protocols, which are often not fully described, have yielded inconsistent CGRP ELISA data in the literature. Here, a modified ELISA protocol for the purification and quantification of CGRP in human plasma is presented. The procedural steps involve sample collection and preparation, extraction using a polar sorbent as a means of purification, additional steps to block non-specific binding, and quantification via ELISA. Further, the protocol has been validated with spike and recovery and linearity of dilution experiments. This validated protocol can theoretically be used to quantify CGRP concentrations in the plasma of individuals not only with migraine, but also with other diseases in which CGRP may play a role.

Detection and Quantification of Calcitonin Gene-Related Peptide CGRP in Human Plasma Using a Modified Enzyme-Linked Immunosorbent Assay

Published data pertaining to calcitonin gene-related peptide (CGRP) concentrations in human plasma are inconsistent. These inconsistencies may be due to the lack of a standardized, validated methodology to quantify this neuropeptide. Here, we describe a validated enzyme-linked immunosorbent assay (ELISA) protocol to purify and quantify CGRP in human plasma.

Calcitonin gene-related peptide (CGRP) is a vasoactive neuropeptide that plays a putative role in the pathophysiology of migraine headaches and may be a ...

Smartphone as an Analytical Device to Quantify Protein Using Bradford Assay

RGBradford: Protein Quantitation with a Smartphone Camera

Protein quantitation is an essential procedure in life sciences research. Amongst several other methods, the Bradford assay is one of the most used. Because of its widespread, the limitations and advantages of the Bradford assay have been exhaustively reported, including several modifications of the original method to improve its performance. One of the alterations of the original method is the use of a smartphone camera as an analytical instrument. Taking advantage of the three forms of the Coomassie Brilliant Blue dye that exist in the conditions of the Bradford assay, this paper describes how to accurately quantify protein in samples using color data extracted from a single picture of a microplate. After performing the assay in a microplate, a picture is taken using a smartphone camera, and RGB color data is extracted from the picture using a free and open-source image analysis software application. Then, the ratio of blue to green intensity (in the RGB scale) of samples with unknown concentrations of protein is used to calculate the protein content based on a standard curve. No significant difference is observed between values calculated using RGB color data and those calculated using conventional absorbance data.

Author Spotlight: An Alternative Approach to Protein Quantification by Bradford Assay Using a Smartphone 

This paper provides a protocol for protein quantification using the Bradford assay and a smartphone as an analytical device. Protein levels in samples can be quantified using color data extracted from a picture of a microplate taken with a smartphone.

Protein quantitation is an essential procedure in life sciences research. Amongst several other methods, the Bradford assay is one of the most used. ...