This study was supported by the National Science Foundation award MCB-2012074. The authors thank Dr. Craig W. Vander Kooi of the University of Florida Department of Biochemistry and Molecular Biology for valuable discussions and support. The authors also thank Dr. Matthew S. Gentry of the University of Florida Department of Biochemistry and Molecular Biology for his support. We would like to thank Dr. Sara Lagalwar, chair of the Skidmore College Neuroscience program, for allowing us to use the LICOR C-digit blot scanner for western blot imaging.&#160;

1. Preparation of ConA-Sepharose beads
<ol>
	<li>Make 250 mL of a binding buffer containing 67 mM HEPES (pH 7.5), 10 mM MgCl2, and 0.2 mM CaCl2. Adjust the pH using 1 M NaOH solution.</li>
	<li>Pipette 250 &#956;L of ConA-Sepharose bead suspension into a 1.5 mL microcentrifuge tube. Centrifuge the contents at 10,000 x g for 30 s at 4 &#176;C. Discard the supernatant. 
	NOTE: 250 &#181;L of ConA-Sepharose beads in a 1.5 mL microcentrifuge tube is needed for each amylopectin concentration used for the assay.</li>
	<li>Add 750 &#181;L of the binding buffer to each tube containing 250 &#181;L of ConA-Sepharose beads. Centrifuge the tubes at 10,000 x g for 1 min at 4 &#176;C. Remove the supernatant. Repeat this step 2x to ensure that the beads are appropriately washed and equilibrated with the binding buffer.</li>
</ol>
2. Preparation of amylopectin solutions
<ol>
	<li>Make a stock solution of 10 mg/mL potato amylopectin. Amylopectin is water-insoluble and can be solubilized by heat. To solubilize, add 0.1 g of potato amylopectin to 10 mL of distilled water. Heat the suspension in a water bath at 80 &#176;C for 1 h or until the solution is no longer cloudy.</li>
	<li>Allow the solution to return to room temperature (RT), with repeated vortexing to avoid clumping.</li>
	<li>Alcohol-alkaline treatment is an alternative method to solubilize amylopectin substrates. To solubilize using this method, follow the steps below.
	<ol>
		<li>Suspend 0.5 g of amylopectin substrate in 5 mL of 20% ethanol and 5 mL of 2 M NaOH. Stir the contents vigorously for 15-20 min at RT.</li>
		<li>Next, add 10 mL of water, and adjust the pH of the solution to 6.5 by adding 2 M HCl. Bring up the volume of the resulting solution to 50 mL with distilled water to make a 10 mg/mL amylopectin solution.</li>
	</ol>
	</li>
	<li>Dilute the 10 mg/mL solubilized amylopectin solution to make a series of 2 mL of diluted amylopectin solutions. For example, perform half dilutions of 10 mg/mL to prepare a series of amylopectin concentrations (5 mg/mL, 2.5 mg/mL, 1.25 mg/mL, 0.625 mg/mL, 0.3125 mg/mL, 0.156 mg/mL, 0.078 mg/mL, 0.039 mg/mL, 0.019 mg/mL, and 0 mg/mL).</li>
</ol>
3. Preparation of ConA-Sepharose: amylopectin beads
<ol>
	<li>Add 250 &#181;L of each diluted amylopectin solution to 1.5 mL microcentrifuge tubes containing 250 &#181;L of ConA-Sepharose beads pre-equilibrated in binding buffer. Mix the contents well. Label the tubes with the corresponding amylopectin concentration.</li>
	<li>Incubate the contents on a rotating wheel at 4 &#176;C for 30 min. 
	NOTE: There is no change in the ConA-Sepharose:amylopectin bound complex over time after 20 min. The 30 min incubation time was chosen by varying incubation times from 10 min to 1 h to ensure equilibrium was reached.</li>
	<li>Centrifuge the tubes at 10,000 x g for 1 min. Collect the supernatant in a newly labeled 1.5 mL microcentrifuge tube. Save these supernatant fractions to perform D-glucose assay12 (acid hydrolysis of amylopectin followed by UV determination of glucose via enzymatic assay). This step is necessary to ensure all amylopectin is bound to the beads.</li>
	<li>Add 750 &#181;L of binding buffer to the ConA-Sepharose:amylopectin beads. Centrifuge the tubes at 10,000 x g for 1 min. Discard the supernatant to remove any unbound amylopectin molecules.</li>
	<li>Repeat step 3.4 to ensure sufficient washing. Each tube now contains ConA-Sepharose beads bound to varying amounts of amylopectin substrates.</li>
</ol>
4. Incubating SEX4 with ConA-Sepharose:amylopectin beads
<ol>
	<li>Mix 250 &#181;L of ConA-Sepharose:amylopectin beads with 100 &#181;L of the binding buffer which includes 10 &#181;g of SEX4 protein, 10 mM dithiothreitol (DTT), and 10 &#181;M protease inhibitor cocktail (PIC). Note that the total volume in each tube is 350 &#181;L. 
	NOTE: A protease inhibitor cocktail is added as a precautionary step to avoid any unnecessary SEX4 degradation. This is an optional step. In this assay, the recombinant protein Arabidopsis thaliana SEX4 (AtSEX4) is used. The purified protein contains an N-terminal histidine tag necessary for detecting the protein via chemiluminescence. Detailed information on glucan phosphatase purifications is described in previous publications14,20,24.</li>
	<li>Incubate the protein and ConA-Sepharose:amylopectin bead suspension at 4 &#176;C for 45 min with gentle rotation. 
	NOTE: The 45 min incubation time is chosen to ensure equilibrium is reached for the complex.</li>
	<li>Centrifuge the tubes at 10,000 x g for 1 min. Pipette 50 &#181;L of the supernatant carefully using a gel loading tip into a new 1.5 mL microcentrifuge tube. Add 20 &#181;L of 4x SDS-PAGE dye and 10 &#181;L of water to each tube containing 50 &#181;L of the collected supernatant fractions. Heat the samples at 95 &#176;C for 10 min. Save these samples for running the SDS-PAGE gels. Ensure 10 new tubes labeled &#34;supernatant (S)&#34; have the corresponding substrate concentrations.</li>
	<li>Add 750 &#181;L of the binding buffer to the ConA-Sepharose:amylopectin: SEX4 beads to remove any unbound protein from the beads. Centrifuge the tubes at 10,000 x g for 1 min. Repeat this step one more time to ensure proper washing. Discard the supernatant.</li>
	<li>Add 20 &#181;L of 4x SDS-PAGE dye and 80 &#181;L of distilled water into the tubes containing washed ConA-Sepharose:amylopectin:SEX4 beads. Heat the samples at 95 &#176;C for 10 min and centrifuge at 10,000 x g for 1 min.</li>
	<li>Discard the pellet and save the supernatant for running the SDS-PAGE gels. Pipette 80 &#181;L of the supernatant into new tubes and label them as &#34;pellet (P)&#34;.</li>
</ol>
5. Running SDS-PAGE gels
<ol>
	<li>Load 40 &#181;L of the unbound protein samples (made in step 2.3, labeled S) into 4%-12% precast polyacrylamide gel wells from the lowest substrate concentration to the highest, but keep the first lane free to load the protein molecular weight marker. Use a second gel to load 10 bound protein samples made in step 2.5 (labeled as P).</li>
	<li>Add freshly prepared 1x SDS-PAGE running buffer to both chambers of the apparatus. Run the gel at 150 V for 35 min or until the dye front reaches the bottom of the gel.</li>
	<li>Remove the run gel from the apparatus and remove the spacers and glass plates. Use the separated gel to run a western blot analysis.</li>
</ol>
6. Western blotting for chemiluminescence detection14,15
NOTE: This method can be easily modified/adapted depending on the western blotting equipment that users have in their labs.
<ol>
	<li>Make 1 L of transfer buffer containing 5.8 g of Tris base, 2.9 g of glycine, 0.37 g of SDS, and 200 mL of methanol.</li>
	<li>Transfer the size-separated proteins from the polyacrylamide gel to a nitrocellulose membrane. Briefly assemble the sponges, filter papers, gel, and nitrocellulose membrane according to western transfer protocol14,15. Run at 70 V for 1 h.</li>
	<li>To prevent nonspecific protein binding, incubatethe nitrocellulose membrane containing the protein solution of 1%-5% bovine serum albumin (BSA) or milk protein in 50 mL of TBST buffer (20 mM Tris [pH 7.5], 150 mM NaCl, 0.1% Tween 20) for 1 h. Wash the membrane 3x using TBST buffer to remove any unbound blocking solution.</li>
	<li>Incubate the membrane with a horseradish peroxidase (HRP)-linked antibody specific for His-tagged protein for 1 h. Wash the membrane 3x in TBST buffer to remove any unbound antibodies. Use a 1:2,000 dilution of antibody to TBST for optimal reproducibility and sensitivity.</li>
	<li>The HRP enzyme-linked antibody binds specifically to the histidine tag of the SEX4 protein, which yields a band in the presence of chemiluminescence reagents. For digital imaging, make a solution of equal parts of chemiluminescent substrate solutions (750 &#181;L each) in a 1.5 mL tube. Incubate the membrane for at least 5 min in the solution.</li>
	<li>Place the membrane protein side down on the blot scanner and run the acquisition software to quantify the protein in both the pellet and supernatant fractions.</li>
</ol>
7. Data analysis
<ol>
	<li>Perform the quantitative signal measurements using the acquisition software with the blot scanner. Normalize all quantitative measurements in the supernatant and pellet fractions to the total protein loaded. 
	NOTE: The software allows to quantify the intensity of each protein band in the supernatant and pellet fractions.</li>
	<li>In the saturating binding experiment, plot the percentage of protein-bound versus amylopectin concentration. Fit the data to Y = Bmax x X/(KD + X), using data analysis software to calculate KD. 
	&#8203;NOTE: Bmax is the maximum specific binding, the Y-axis is the percentage of protein bound, the and X-axis is the amylopectin concentration.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64700/64700fig01.jpg" /> 
Figure 1: Overview of the ConA-Sepharose sedimentation assay workflow. (A)&#160;Preparation of ConA-Sepharose beads. (B) Incubation with amylopectin substrate. (C) Incubation with SEX4 protein. (D) Separation of bound and unbound protein fractions through centrifugation. (E) Separation of protein through SDS-PAGE. (F) Western-blot analysis. (G) Chemiluminescence detection of His-tagged SEX4 protein. <a href="https://www.jove.com/files/ftp_upload/64700/64700fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laforin,+a+dual+specificity+phosphatase+that+dephosphorylates+complex+carbohydrates.">Laforin, a dual specificity phosphatase that dephosphorylates complex carbohydrates.</a> The Journal of Biological Chemistry. 281 (41), 30412-30418 (2006).</li><li>Gentry, M. S., Pace, R. 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Involvement of carbohydrate recognition and hydrophobic interaction.</a> Biochemistry. 15 (3), 704-713 (1976).</li><li>Meekins, D. 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The authors declare no conflicts of interest.

This study demonstrates the successful development of a novel in vitro sedimentation assay that allows determination of the binding affinity of glucan-glucan phosphatase interactions. The assay design takes advantage of the specific binding of lectin ConA to glucans via the hydroxyl residues of glucose to indirectly capture solubilized carbohydrate substrates onto Sepharose beads. This allows the separation of bound and unbound protein fractions via centrifugation and determination of the bindi...

Glucan phosphatases are members of a functionally diverse subfamily of dual specificity phosphatases (DSPs) within the protein tyrosine phosphatase (PTP) superfamily1. They have been found in most life forms, including widely divergent photosynthetic organisms, humans, vertebrates, and some invertebrates and protists2,3,4. Plants contain three known glucan phosphatases: Starch Excess4 (SEX4), Like Sex Four1 (LSF1), and Like Sex Four2 (LSF2)5,6,7. Plants that lack glucan phosphatases display decreased rates of transitory starch degradation and accumulation of starch in the leaves8,9. Laforin is the founding member of the glucan phosphatase family that dephosphorylates glycogen in vertebrates and humans3,10. The mutations of laforin result in neurodegenerative Lafora disease, a fatal autosomal recessive form of epilepsy11. Glucan phosphatases are necessary for glycogen and starch metabolism and have emerged as important enzymes for modulating starch content in plants and treating neurodegenerative Lafora disease12,13. Recent X-ray crystallography studies on glucan phosphatases with model glucan substrates have shed light on substrate binding and the catalytic mechanism of glucan dephosphorylation14,15,16,17. However, the current understanding of how glucan phosphatases bind to their physiological substrates is incomplete.
Starch is an insoluble polymer of glucose made of 80%-90% amylopectin and 10%-20% amylose18. The substrates for plant glucan phosphatases are phosphorylated carbohydrate molecules, such as glycogen and starch granules. The phosphorylated glucosyl residues are present at a 1:600 phosphate:glucosyl residue ratio. Interestingly, the phosphates are present only on the amylopectin molecules19. The main plant glucan phosphatase SEX4 acts on the starch granule to dephosphorylate amylopectin molecules. The X-ray crystal structure of SEX4 combined with structure-guided mutagenesis studies has demonstrated the unique substrate specificities of SEX4 for different positions within a glucan structure15. We recently showed that the biologically relevant activity of SEX4 can only be observed when acting on its solubilized amylopectin substrates20. However, understanding glucan-SEX4 interactions has proven to be difficult due to the structural complexity of the substrate, broader binding specificities, and low binding affinities between the protein and its substrates. These issues have hindered the ability to utilize methods commonly used in protein-ligand interactions, such as isothermal titration calorimetry (ITC), nuclear magnetic resonance (NMR) spectroscopy, and enzyme-linked immunosorbent assay (ELISA)-based assays.
Interestingly, much of our understanding of carbohydrate-protein interactions have come from studying lectins. Concanavalin A (ConA) is a legume lectin family of proteins originally extracted from the jack bean. ConA binds carbohydrates with high specificity, which is advantageous for its use in drug targeting and delivery applications. The binding of ConA to a variety of substrates containing nonreducing &#945;-D-mannosyl and &#945;-D-glucosyl has been extensively studied19,20. Commercially available ConA-bound Sepharose beads are commonly used to purify glycoproteins and glycolipids21. ConA binds to these glucans via C3, C4, and C6 hydroxyl groups of the glucose residues. ConA-Sepharose beads have also been successfully used to measure the binding of glycogen-protein and starch-protein interactions22,23. In this study, we used ConA-Sepharose beads to develop a binding assay to measure the binding specificities of glucan phosphatase-amylopectin interactions.
Previously, a ConA-based sedimentation assay was employed to assess glucan phosphatase substrate binding ability14,20,24. In this study, the same strategy was used to develop a novel method to determine the binding affinity of glucan-glucan phosphatase and carbohydrate interactions. This method also has an advantage for investigating various solubilized carbohydrate-protein interactions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6x-His Tag monoclonal antibody (HIS.H8), HRP</td>
			<td>Therm Fisher Scientific</td>
			<td>MA1-21315-HRP</td>
			<td></td>
		</tr>
		<tr>
			<td>Biorad gel electrophoresis and Western blot kit</td>
			<td>Biorad&#160;</td>
			<td>1703930</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>208291</td>
			<td></td>
		</tr>
		<tr>
			<td>C-Digit blot scanner</td>
			<td>LICOR</td>
			<td>3600-00</td>
			<td>Blot scanner</td>
		</tr>
		<tr>
			<td>Complete protease inhibitor cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>11836170001</td>
			<td></td>
		</tr>
		<tr>
			<td>Concanavalin A-sepharose beads</td>
			<td>Sigma-Aldrich</td>
			<td>C9017</td>
			<td>This product contains&#160; in 0.1 M acetate buffer, pH 6, containing 1 M NaCl, 1 mM CaCl2, 1 mM MnCl2, and 1 mM MgCl2 in 20% ethanol&#160;</td>
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			<td>Westernsure premium chemiluminescence substrate&#160;</td>
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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.

One of the key features of the glucan phosphatase family of proteins is their ability to bind to glucan substrates. First, the binding capacity of SEX4 to ConA-Sepharose:amylopectin beads was analyzed using SDS-PAGE (Figure 2A). Bovine serum albumin (BSA) served as a negative control to detect any nonspecific binding of proteins to the ConA-Sepharose:amylopectin beads. The SDS-PAGE analysis of proteins showed the presence of SEX4 protein in the pellet fraction and BSA in the supernatant frac...

Watch this Scientific Journal Video about Concanavalin A-Based Sedimentation Assay to Measure Substrate Binding of Glucan Phosphatases at JoVE.com

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

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 ...

concanavalin-based-sedimentation-assay-to-measure-substrate-binding

Research

JoVE Journal

Biochemistry

1.2K Views.  Skidmore College. 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. 

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

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper and food industries. A deeper understanding of CAZymes is important from both fundamental biology and industrial standpoints. Vast numbers of CAZymes exist in nature (especially in microorganisms) and hundreds of thousands have been cataloged and described in the carbohydrate active enzyme database (CAZy). However, the rate of discovery of putative enzymes has outstripped our ability to biochemically characterize their activities. One reason for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay kit based on insoluble chromogenic substrates is described here. Two distinct substrate types were produced: Chromogenic Polymer Hydrogel (CPH) substrates (made from purified polysaccharides and proteins) and Insoluble Chromogenic Biomass (ICB) substrates (made from complex biomass materials). Both CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used for screening the activities of enzymes, enzyme cocktails, and broths.

A high-throughput assay for enzyme screening is described. This multiplexed ready-to-use assay kit comprises of pre-chosen Chromogenic Polymer Hydrogel (CPH) substrates and complex Insoluble Chromogenic Biomass (ICB) substrates. Target enzymes are polysaccharide degrading endo-enzymes and proteases.

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay

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

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

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

Measuring Protein Binding to F-actin by Co-sedimentation

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, cross-linking and/or disassembly. This protocol describes a technique &#8211; the actin co-sedimentation, or pelleting, assay &#8211; to determine whether a protein or protein domain binds F-actin and to measure the affinity of the interaction (i.e., the dissociation equilibrium constant). In this technique, a protein of interest is first incubated with F-actin in solution. Then, differential centrifugation is used to sediment the actin filaments, and the pelleted material is analyzed by SDS-PAGE. If the protein of interest binds F-actin, it will co-sediment with the actin filaments. The products of the binding reaction (i.e., F-actin and the protein of interest) can be quantified to determine the affinity of the interaction. The actin pelleting assay is a straightforward technique for determining if a protein of interest binds F-actin and for assessing how changes to that protein, such as ligand binding, affect its interaction with F-actin.

This protocol describes a method to test the ability of a protein to co-sediment with filamentous actin (F-actin) and, if binding is observed, to measure the affinity of the interaction.

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, ...

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 ...

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant strains. Carbohydrate substrate binding proteins (SBPs) represent viable targets for the development of protein-based vaccines and new antimicrobials because of their extracellular localization and the centrality of carbohydrate import for pneumococcal metabolism, respectively. Described here is a rationalized integrated protocol to carry out a comprehensive characterization of SP0092, which can be extended to other carbohydrate SBPs from the pneumococcus and other bacteria. This procedure can aid the structure-based design of inhibitors for this class of proteins. Presented in the first part of this manuscript are protocols for biochemical analysis by thermal shift assay, multi angle light scattering (MALS), and size exclusion chromatography (SEC), which optimize the stability and homogeneity of the sample directed to crystallization trials and so enhance the probability of success. The second part of this procedure describes the characterization of the SBP crystals using a tunable wavelength anomalous diffraction synchrotron beamline, and data collection protocols for measuring data that can be used to resolve the crystallized protein structure.

A streamlined protocol for performing an extensive biochemical and structural characterization of a carbohydrate substrate binding protein from Streptococcus pneumoniae is presented.

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant ...

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 ...

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

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

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

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

A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors

Most cellular processes are regulated by dynamic protein phosphorylation. More than three-quarters of proteins are phosphorylated, and phosphoprotein phosphatases (PPPs) coordinate over 90% of all cellular serine/threonine dephosphorylation. Deregulation of protein phosphorylation has been implicated in the pathophysiology of various diseases, including cancer and neurodegeneration. Despite their widespread activity, the molecular mechanisms controlling PPPs and those controlled by PPPs are poorly characterized. Here, a proteomic approach termed phosphatase inhibitor beads and mass spectrometry (PIB-MS) is described to identify and quantify PPPs, their posttranslational modifications, and their interactors in as little as 12 h using any cell line or tissue. PIB-MS utilizes a non-selective PPP inhibitor, microcystin-LR (MCLR), immobilized on sepharose beads to capture and enrich endogenous PPPs and their associated proteins (termed the PPPome). This method does not require the exogenous expression of tagged versions of PPPs or the use of specific antibodies. PIB-MS offers an innovative way to study the evolutionarily conserved PPPs and expand our current understanding of dephosphorylation signaling.

Here, we present a protocol for the enrichment of endogenous phosphoprotein phosphatases and their interacting proteins from cells and tissues and their identification and quantification by mass spectrometry-based proteomics.

Most cellular processes are regulated by dynamic protein phosphorylation. More than three-quarters of proteins are phosphorylated, and phosphoprotein ...

A Fibrinolytic Enzyme-Based Method to Study Cerebral Thrombus Cell Components

A Comprehensive Approach to Analyze the Cell Components of Cerebral Blood Clots

Cerebral thrombosis, a blood clot in a cerebral artery or vein, is the most common type of cerebral infarction. The study of the cell components of cerebral blood clots is important for diagnosis, treatment, and prognosis. However, the current approaches to studying the cell components of the clots are mainly based on in situ staining, which is unsuitable for the comprehensive study of the cell components because cells are tightly wrapped in the clots. Previous studies have successfully isolated a fibrinolytic enzyme (sFE) from Sipunculus nudus, which can degrade the cross-linked fibrin directly, releasing the cell components. This study established a comprehensive method based on the sFE to study the cell components of cerebral thrombus. This protocol includes clot dissolving, cell releasing, cell staining, and routine blood examination. According to this method, the cell components could be studied quantitatively and qualitatively. The representative results of experiments using this method are shown.

Author Spotlight: A Novel Method for Comprehensive Cell Component Analysis of Cerebral Blood Clots 

This study describes a fast and effective method for the cell component analysis of cerebral blood clots through clot dissolving, cell staining, and routine blood examination.

Cerebral thrombosis, a blood clot in a cerebral artery or vein, is the most common type of cerebral infarction. The study of the cell components of ...

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

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

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

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

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

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