The work is supported by an NNF Project Grant (NNF19OC0058331) to YEZ, and the European Union&#8217;s Horizon 2020 research and innovation programme under the Marie Sk&#322;odowska-Curie grant agreement (N&#186; 801199) to MLS.
<img alt="Equation 1" src="/files/ftp_upload/62331/62331img1.jpg" />

1. Preparation of whole cell lysates
<ol>
	<li>Inoculate the E. coli K-12 ASKA ORFeome collection strains15 into 1.5 mL Lysogeny broth (LB) containing 25 &#181;g/mL chloramphenicol in 96-well deep well plates. Grow overnight (O/N) for 18 h at 30 &#176;C with shaking at 160 rpm. On the next day, add isopropyl &#946;-d-1-thiogalactopyranoside (IPTG) (final 0.5 mM) to the O/N cultures to induce protein expression at 30 &#176;C for 6 h.</li>
	<li>Pellet cells at 500 x g for 10 min. Freeze the pellets at -80 &#176;C until use. To lyse the cells, add 150 &#181;L of lysis buffer L1 (40 mM Tris pH 7.5, 100 mM NaCl, 10 mM MgCl2, supplemented with 2 mM phenylmethylsulfonyl fluoride (PMSF), 40 &#181;g/mL DNase 1, and 0.5 mg/mL lysozyme) to resuspend the pellet.</li>
	<li>Freeze the cells at -80 &#176;C for 30 min, and then thaw at 37 &#176;C for 20 min. Repeat this cycle three times to lyse the cells. Store the lysates at -80 &#176;C before use.</li>
</ol>
2. Purification of Relseq and GppA
NOTE: The recombinant proteins Relseq from Streptococcus equisimilis and GppA from E. coli K-12 are used to synthesize the radiolabeled pppGpp and ppGpp, respectively.
<ol>
	<li>Grow and collect cells overexpressing each protein.
	<ol>
		<li>Grow the E. coli BL21 DE3 strain up to exponential phase (optical density (OD) ~0.3-0.4) in LB broth, and spin down 1 mL of culture at 6000 x g for 5 min. Decant the supernatant, and resuspend the cells with 100 &#181;L of ice-cold TSB broth (LB broth supplemented with 0.1 g/mL PEG3350, 0.05 mL/mL dimethylsulfoxide, 20 mM MgCl2).</li>
		<li>Mix the plasmids bearing the histidine-tagged relseq and gppA, each 100 ng, with the above cell suspensions in TSB, and incubate on ice for 30 min. Heat-shock the cells at 42 &#176;C for 40 s. Place the mixture on ice for 2 min, and add 1 mL of LB broth at room temperature to allow the cells to recover for 1 h at 37 &#176;C with agitation at 160 rpm.</li>
		<li>Plate the recovered cells on LB agar plates supplemented with the corresponding antibiotics (Relseq:100 &#181;g/mL ampicillin; GppA: 30 &#181;g/mL kanamycin).On the next day, inoculate the colonies in LB broth to start O/N precultures of both strains at 37 &#176;C.</li>
		<li>After 18 h, inoculate 500 mL of LB medium with 10 mL of the O/N cultures and the corresponding antibiotics. Grow the cultures by shaking at 160 rpm at 37 &#176;C. When the OD600nm reaches 0.5-0.7, induce protein expression by adding 0.5 mM IPTG and growing for 3 h at 30 &#176;C with shaking at 160 rpm.</li>
		<li>Collect the cells by spinning at 6084 x g for 10 min at 4 &#176;C. Resuspend the pellet in 20 mL of ice-cold 1x phosphate-buffered saline (PBS), and re-centrifuge at 1912 x g for 20 min at 4 &#176;C. Decant the supernatant, and freeze the pellets at -20 &#176;C before use.</li>
	</ol>
	</li>
	<li>Nickel-nitrilotriacetic acid (Ni-NTA) affinity purification 
	&#8203;NOTE: From this point onwards, ensure that the samples are cold.
	<ol>
		<li>Add 40 mL of ice-cold lysis buffer L2 (50 mM Tris pH 7.5, 150 mM NaCl, 5% glycerol, 10 mM imidazole, 10 mM &#946;-mercaptoethanol supplemented with protease inhibitors (EDTA-free tablet; see the Table of Materials) to resuspend the pellet. Lyse the cells via sonication (60% amplitude, 2 s ON/ 4 s OFF for 8 min ON). Clear the lysate by spinning at 23,426 x g for 40 min at 4 &#176;C, and continue with the supernatant for the purification.</li>
		<li>During the above centrifugation, prepare the Ni-NTA resin.
		<ol>
			<li>Transfer 500 &#181;L of homogenized Ni-NTA resin into a standing polypropylene chromatography column, and let it settle for 15 min and the storage solution drain through. Wash the resin with 15 mL of ultrapure water twice, and then wash the column with 15 mL of the lysis buffer L2.</li>
		</ol>
		</li>
		<li>Load the cleared supernatant of cell lysate from step 1 onto the column, and let it flow through. Wash the column with 30 mL of washing buffer (50 mM Tris pH 7.5, 150 mM NaCl, 5% glycerol, 20 mM imidazole).</li>
		<li>Elute the proteins with 400 &#181;L of the elution buffer (50 mM Tris pH 7.5, 150 mM NaCl, 5% glycerol, 500 mM imidazole) three times. Then, repeat elution with another 300 &#181;L of the elution buffer. Combine the eluted proteins to a final volume of 700 &#181;L.</li>
	</ol>
	</li>
	<li>Gel filtration
	<ol>
		<li>Prepare gel filtration buffer (50 mM Tris, pH 7.5; 200 mM NaCl; 5% glycerol). Wash the size exclusion column with one column volume (25 mL) of the gel filtration buffer.</li>
		<li>Load the above 700 &#181;L sample by using a 500 &#181;L loop, run at 0.5 mL/min, and collect 2-3 fractions, each of 0.5 mL volume, containing the respective proteins.</li>
		<li>Combine and concentrate the fractions containing each of the proteins using a spin column, and measure the protein concentration using the Bradford assay.</li>
	</ol>
	</li>
</ol>
3. Synthesis of 32P-labeled pppGpp and ppGpp
<ol>
	<li>Assemble a small-scale Relseq reaction in a screw cap tube (see Table 1). 
	NOTE: Work with radioactive reagents only in a licensed place and with personal protective equipment.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="2">Volume (&#956;L)</td>
		</tr>
		<tr>
			<td></td>
			<td>Small scale</td>
			<td>Large scale</td>
		</tr>
		<tr>
			<td>Ultrapure water</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10x Relseq buffer*</td>
			<td>2</td>
			<td>50</td>
		</tr>
		<tr>
			<td>ATP (8 mM final)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Relseq (4 &#956;M final)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>32P-&#945;-Guanosine triphosphate (GTP) (final 120 nM) (CAUTION)</td>
			<td>0.2</td>
			<td>5</td>
		</tr>
		<tr>
			<td>total&#160;</td>
			<td>20</td>
			<td>500</td>
		</tr>
	</tbody>
</table>
Table 1: Assembling information for the small- and large-scale synthesis reactions of 32P-labeled pppGpp. *10x Relseq buffer contains 250 mM Tris-HCl, pH 8.6; 1M NaCl; 80 mM MgCl2. Abbreviation: pppGpp = guanosine pentaphosphate.
<ol start="2">
	<li>Incubate the tube at 37 &#176;C in a thermomixer for 1 h, then at 95 &#176;C for 5 min, and place on ice for 5 min. Spin down the precipitated protein at 15,700 x g for 5 min, and transfer the supernatant (synthesized 32P-pppGpp) to a new screw cap tube.</li>
	<li>To synthesize 32P-ppGpp from 32P-pppGpp, transfer half of the 32p-pppGpp product to a new screw cap tube, and add 1 &#181;M GppA. Incubate the tube at 37 &#176;C for 10 min, at 95 &#176;C for 5 min, and then place on ice for 5 min.</li>
	<li>Spin down the precipitated protein at 15,700 x g for 5 min, and transfer the supernatant (synthesized 32P-ppGpp) to a new screw cap tube.</li>
	<li>Analyze the 32P-pppGpp and 32P-ppGpp by running 1 &#181;L of the samples on a thin layer chromatography (TLC) plate (polyethyleneimine-modified cellulose TLC plates) using the 1.5 M KH2PO4, pH 3.4, as mobile phase. 
	NOTE: Use the &#945;-32P-labeled guanosine 5&#39;-triphosphate (32P-&#945;-GTP) as control.</li>
	<li>Dry the TLC plate completely, place it between a transparent plastic folder, and expose it to a storage phosphor screen for 5 min. Visualize and quantify the signals by using a phosphorimager. 
	&#8203;NOTE: When the ratios of 32P-pppGpp and 32P-ppGpp are higher than 85%, a large-scale reaction (500 &#181;L, sufficient for screening 20 96-well plates) could be assembled and synthesized by using Table 1.</li>
</ol>
4. DRaCALA screening of the target proteins of (p)ppGpp
<ol>
	<li>Thaw and transfer 20 &#181;L of whole cell lysates to a 96-well V-bottom microtiter plate. Add 2.5 U/well of endonuclease from Serratia marcescens, and incubate at 37 &#176;C for 15 min to reduce lysate viscosity. Place the lysates on ice for 20 min.</li>
	<li>Mix the 32P-pppGpp and 32P-ppGpp in a 1:1 ratio, and add 1x lysis buffer L1 to make the final concentration of (p)ppGpp equal to 4 nM. 
	NOTE: Given the chemical similarity between pppGpp and ppGpp, a mix of both chemicals will simplify the screening process.</li>
	<li>Use a multichannel pipette and filtered pipette tips to add and mix 10 &#181;L of the (p)ppGpp mixture with the cell lysate. Incubate at room temperature (RT) for 5 min.</li>
	<li>Wash the 96 x pin tool by placing in 0.01% solution of non-ionic detergent for 30 s, and dry on a tissue paper for 30 s. Repeat the washing of the pin tool 3x.</li>
	<li>Place the pin tool in the above 96-well sample plates, and wait for 30 s. Lift the pin tool straight up, and place it straight down on a nitrocellulose membrane for 30 s. 
	&#8203;NOTE: If a spot is missing, spot 2 &#181;L of the corresponding samples with a pipette and filtered tips. It is advisable to make a duplicate spot of the same sample as indicated below.</li>
	<li>Dry the membrane for 5 min at RT. Place the membrane between a transparent plastic folder, and expose it to a storage phosphor screen for 5 min. Visualize by using a phosphorimager.</li>
</ol>
5. Quantification and identification of potential target proteins
<ol>
	<li>Use the analysis software associated with the phosphorimager to open the .gel file of the visualized plates. Use the Array analysis function to define the 96 spots by setting up a grid of 12 columns x 8 rows.</li>
	<li>Define big circles to circumscribe the outer edge of the whole spots (see Figure 1B). Export the Volumn+Background and Area of the defined big circles, and save in a spreadsheet. 
	NOTE: If required, reposition each individual circle to perfectly overlap with the spots, and resize each individual circle to make it slightly bigger than the actual spot.</li>
	<li>Size down the defined circles to circumscribe the small inner dots. Export the Volumn+Background, and Area of the defined small circles, and save in a spreadsheet.</li>
	<li>Calculate the binding fractions in the spreadsheet by using the equation in Figure 1B, and plot the data. Identify the potential binding proteins in the wells that show high binding fractions in comparison to the majority of other wells.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62331/62331fig02.jpg" /> 
Figure 2:&#160;Overall workflow of the DRaCALA screening process. Protein production from an Escherichia coli ASKA collection is induced, and the cells are lysed. Meanwhile, the recombinant proteins Relseq-His and GppA-His are purified and used to synthesize 32P-labeled pppGpp and ppGpp from 32P-&#945;-GTP. The radioactively labeled (p)ppGpp molecules are then mixed with the lysates, and a 96 pin-tool is used to spot the mixtures onto a nitrocellulose membrane for subsequent exposure to a phosphor storage screen, imaging, and quantification of the radioactive signals. Abbreviations: DRaCALA = Differential Radial Capillary Action of Ligand Assay; (p)ppGpp = guanosine penta- and tetraphosphates; RT = room temperature; IPTG = isopropyl &#946;-d-1-thiogalactopyranoside; GTP = guanosine 5&#39;-triphosphate; SDS-PAGE = sodium dodecylsulfate-polyacrylamide gel electrophoresis ; TLC = thin layer chromatography. <a href="https://www.jove.com/files/ftp_upload/62331/62331fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The authors have no conflict of interest to disclose.

One of the critical steps in performing DRaCALA screening is to obtain good whole cell lysates. First, the tested proteins should be produced in large amounts and in soluble forms. Second, the lysis of cells should be complete, and the viscosity of the lysate must be minimal. The inclusion of lysozyme and the use of three cycles of freeze-thaw are often enough to lyse cells completely. However, the released chromosomal DNA makes the lysate viscous and generates high background binding signal, resulting in false positives...

Bacteria use several small signaling molecules to adapt to constantly changing environments1,2. For example, the autoinducers, N-acylhomoserine lactones and their modified oligopeptides, mediate the intercellular communication among bacteria to coordinate population behavior, a phenomenon known as quorum sensing2. Another group of small signaling molecules is the NSMs, including the widely studied cyclic adenosine monophosphate (cAMP), cyclic di-AMP, cyclic di-guanosine monophosphate (cyclic di-GMP), and guanosine penta- and tetra phosphates (p)ppGpp1. Bacteria produce these NSMs as a response to a variety of different stress conditions. Once produced, these molecules bind to their target proteins and regulate several different physiological and metabolic pathways to cope with the encountered stresses and enhance bacterial survival. Therefore, identification of the target proteins is an unavoidable prerequisite for deciphering the molecular functions of these small molecules.
The past decade has witnessed a boom of knowledge of these small signaling molecules, mainly due to several technical innovations that unveiled the target proteins of these small molecules. These include the capture compound technique3,4,5, and the differential radial capillary action of ligand assay (DRaCALA)6 to be discussed in this paper.
Invented by Vincent Lee and co-workers in 20116, DRaCALA deploys the ability of a nitrocellulose membrane to differentially sequester free and protein-bound ligands. Molecules such as proteins cannot diffuse on a nitrocellulose membrane, while small ligands, such as the NSMs, are able to. By mixing the NSM (e.g., ppGpp) with the protein to be tested and spotting them on the membrane, two scenarios can be expected (Figure 1): If (p)ppGpp binds to the protein, the radiolabeled (p)ppGpp will be retained in the center of the spot by the protein and will not diffuse outward, giving an intense small dot (i.e., strong radioactive signal) under a phosphorimager. However, if (p)ppGpp does not bind to the protein, it will diffuse freely outward to produce a large spot with uniform background radioactive signal.
Furthermore, DRaCALA can detect the interaction between a small molecule and an unpurified protein in a whole cell lysate if the protein is present in a sufficient amount. This simplicity allows the use of DRaCALA in rapidly identifying protein targets by using an ORFeome expression library. Indeed, target proteins of cAMP7, cyclic di-AMP8, cyclic di-GMP9,10, and (p)ppGpp11,12,13 have been systematically identified by using DRaCALA. This video article uses (p)ppGpp as an example to demonstrate and describe the critical steps and considerations in performing a successful DRaCALA screening. Of note, a more thorough description of DRaCALA14 is highly recommended to read in combination with this article before performing DRaCALA.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62331/62331fig01.jpg" /> 
Figure 1:&#160;The principle of DRaCALA. (A) Schematic of the DRaCALA assay. See the text for details. (B) Quantification and calculation of the binding fraction. See the text for details. Briefly, the DRaCALA spots will be analyzed by drawing two circles that circumscribe the whole spot and the inner dark dot (i.e., the retained (p)ppGpp due to the binding of the tested protein). The specific binding signal is the radioactive signal of the inner circle (S1) after subtracting the non-specific background signal (calculated by A1 &#215; ((S2-S1)/(A2-A1))). The binding fraction is the specific binding signal divided by the total radioactive signal (S2). Abbreviations: DRaCALA = Differential Radial Capillary Action of Ligand Assay; (p)ppGpp = guanosine penta- and tetraphosphates; RT = room temperature. <a href="https://www.jove.com/files/ftp_upload/62331/62331fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>32P-&#945;-GTP</td>
			<td>Perkinelmer</td>
			<td>BLU006X250UC</td>
			<td></td>
		</tr>
		<tr>
			<td>96 x pin tool</td>
			<td>V&#38;P Scientific</td>
			<td>VP 404</td>
			<td>96 Bolt Replicator, on 9 mm centers, 4.2 mm Bolt Diameter, 24 mm long</td>
		</tr>
		<tr>
			<td>96-well V-bottom microtiter plate</td>
			<td>Sterilin</td>
			<td>MIC9004</td>
			<td>Sterilin Microplate V Well 611V96</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>OXOID - Thermo Fisher</td>
			<td>LP0011</td>
			<td>Agar no. 1</td>
		</tr>
		<tr>
			<td>ASKA collection strain</td>
			<td>NBRP, SHIGEN, JAPAN</td>
			<td></td>
			<td>Ref: DNA Research, Volume 12, Issue 5, 2005, Pages 291&#8211;299. https://doi.org/10.1093/dnares/dsi012</td>
		</tr>
		<tr>
			<td>Benzonase</td>
			<td>SIGMA</td>
			<td>E1014-25KU</td>
			<td>genetically engineered endonuclease from Serratia marcescens</td>
		</tr>
		<tr>
			<td>Bradford Protein Assay Dye</td>
			<td>Bio-Rad</td>
			<td>5000006</td>
			<td>Reagent Concentrate</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>SIGMA</td>
			<td>D8418</td>
			<td>&#8805;99.9%</td>
		</tr>
		<tr>
			<td>DNase 1</td>
			<td>SIGMA</td>
			<td>DN25-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>gel filtration10x300 column</td>
			<td>GE Healthcare</td>
			<td>28990944</td>
			<td>contains 20% ethanol as preservative</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>PanReac AppliChem</td>
			<td>122329.1214</td>
			<td>Glycerol 87% for analysis</td>
		</tr>
		<tr>
			<td>Hypercassette</td>
			<td>Amersham</td>
			<td>RPN 11647</td>
			<td>20 x 40 cm</td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>SIGMA</td>
			<td>56750</td>
			<td>puriss. p.a., &#8805; 99.5% (GC)</td>
		</tr>
		<tr>
			<td>IP Storage Phosphor Screen</td>
			<td>FUJIFILM</td>
			<td>28956474</td>
			<td>BAS-MS 2040 20x 40 cm</td>
		</tr>
		<tr>
			<td>Isopropyl &#946;-d-1-thiogalactopyranoside (IPTG)</td>
			<td>SIGMA</td>
			<td>I6758</td>
			<td>Isopropyl &#946;-D-thiogalactoside</td>
		</tr>
		<tr>
			<td>Lysogeny Broth (LB)</td>
			<td>Invitrogen - Thermo Fisher</td>
			<td>12795027</td>
			<td>Miller&#39;s LB Broth Base</td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>SIGMA</td>
			<td>L4949</td>
			<td>from chicken egg white; BioUltra, lyophilized powder, &#8805;98%</td>
		</tr>
		<tr>
			<td>MgCl2 (Magnesium chloride)</td>
			<td>SIGMA</td>
			<td>208337</td>
			<td></td>
		</tr>
		<tr>
			<td>MilliQ water</td>
			<td></td>
			<td></td>
			<td>ultrapure water</td>
		</tr>
		<tr>
			<td>multichannel pipette</td>
			<td>Thermo Scientific</td>
			<td>4661110</td>
			<td>F1 - Clip Tip; 1-10 ul, 8 x channels</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>VWR Chemicals</td>
			<td>27810</td>
			<td>AnalaR NORMAPUR, ACS, Reag. Ph. Eur.</td>
		</tr>
		<tr>
			<td>Ni-NTA Agarose</td>
			<td>Qiagen</td>
			<td>30230</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose Blotting Membrane</td>
			<td>Amersham Protran</td>
			<td>10600003</td>
			<td>Premium 0.45 um 300 mm x 4 m</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>OXOID - Thermo Fisher</td>
			<td>BR0014G</td>
			<td>Phosphate buffered saline (Dulbecco A), Tablets</td>
		</tr>
		<tr>
			<td>PEG3350 (Polyethylene glycol 3350)</td>
			<td>SIGMA</td>
			<td>202444</td>
			<td></td>
		</tr>
		<tr>
			<td>phenylmethylsulfonyl fluoride (PMSF)</td>
			<td>SIGMA</td>
			<td>93482</td>
			<td>Phenylmethanesulfonyl fluoride solution - 0.1 M in ethanol (T)</td>
		</tr>
		<tr>
			<td>Phosphor-imager</td>
			<td>GE Healthcare</td>
			<td>28955809</td>
			<td>Typhoon FLA-7000 Phosphor-imager</td>
		</tr>
		<tr>
			<td>Pipette Tips, filtered</td>
			<td>Thermo Scientific</td>
			<td>94410040</td>
			<td>ClipTip 12.5 &#956;l nonsterile</td>
		</tr>
		<tr>
			<td>Poly-Prep Chromatography column</td>
			<td>Bio-Rad</td>
			<td>7311550</td>
			<td>polypropylene chromatography column</td>
		</tr>
		<tr>
			<td>Protease inhibitor Mini</td>
			<td>Pierce</td>
			<td>A32955</td>
			<td>Tablets, EDTA-free</td>
		</tr>
		<tr>
			<td>screw cap tube</td>
			<td>Thermo Scientific</td>
			<td>3488</td>
			<td>Microcentifuge Tubes, 2.0 ml with screw cap, nonsterile</td>
		</tr>
		<tr>
			<td>SLS 96-deep Well plates</td>
			<td>Greiner</td>
			<td>780285</td>
			<td>MASTERBLOCK, 2 ML, PP, V-Bottom, Natural</td>
		</tr>
		<tr>
			<td>spin column</td>
			<td>Millipore</td>
			<td>UFC500396</td>
			<td>Amicon Ultra -0.5 ml Centrifugal Filters</td>
		</tr>
		<tr>
			<td>Thermomixer</td>
			<td>Eppendorf</td>
			<td>5382000015</td>
			<td>Thermomixer C</td>
		</tr>
		<tr>
			<td>TLC plate (PEI-cellulose F TLC plates)</td>
			<td>Merck Millipore</td>
			<td>105579</td>
			<td>DC PEI-cellulose F (20 x 20 cm)</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>SIGMA</td>
			<td>BP152</td>
			<td>Tris Base for Molecular Biology</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>SIGMA</td>
			<td>P1379</td>
			<td>viscous non-ionic detergent</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>SIGMA</td>
			<td>M3148</td>
			<td>99% (GC/titration)</td>
		</tr>
	</tbody>
</table>

identifying the binding proteins of small ligands with the differential radial capillary action of ligand assay (dracala)

The past decade has seen tremendous progress in the understanding of small signaling molecules in bacterial physiology. In particular, the target proteins of several nucleotide-derived secondary messengers (NSMs) have been systematically identified and studied in model organisms. These achievements are mainly due to the development of several new techniques including the capture compound technique and the differential radial capillary action of ligand assay (DRaCALA), which were used to systematically identify target proteins of these small molecules. This paper describes the use of the NSMs, guanosine penta- and tetraphosphates (p)ppGpp, as an example and video demonstration of the DRaCALA technique. Using DRaCALA, 9 out of 20 known and 12 new target proteins of (p)ppGpp were identified in the model organism, Escherichia coli K-12, demonstrating the power of this assay. In principle, DRaCALA could be used for studying small ligands that can be labeled by radioactive isotopes or fluorescent dyes. The critical steps, pros, and cons of DRaCALA are discussed here for further application of this technique.

Following the above-described protocol will typically yield two types of results (Figure 3).
Figure 3A shows a plate with relatively low background binding signals (binding fractions &#60; 0.025) from the majority of wells. The positive binding signal from the well H3 gives a binding fraction of ~0.35 that is much higher than that observed for the other wells. Even without quantification, well H3 is remarkable, suggesting that a targe...

Watch this Scientific Journal Video about Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay DRaCALA at JoVE.com

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay DRaCALA

The Differential Radial Capillary Action of Ligand Assay (DRaCALA) can be used to identify small ligand binding proteins of an organism by using an ORFeome library.

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay (DRaCALA)

The past decade has seen tremendous progress in the understanding of small signaling molecules in bacterial physiology. In particular, the target proteins ...

identifying-binding-proteins-small-ligands-with-differential-radial

Research

JoVE Journal

Biochemistry

3.4K Views.  University of Copenhagen. The Differential Radial Capillary Action of Ligand Assay (DRaCALA) can be used to identify small ligand binding proteins of an organism by using an ORFeome library.

Video: Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay DRaCALA

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several target identification methods have been developed and used to successfully elucidate the binding proteins of a variety of small molecules, these techniques have drawbacks that make them unsuitable for detecting certain types of small molecule-target interactions. In particular, non-covalent interactions that depend on native cellular conditions, such as those of membrane proteins whose structures may be perturbed upon cell lysis, are often not amenable to affinity-based target identification methods. Here, we demonstrate a method wherein a probe containing a photolabile group is used to covalently crosslink to the small molecule binding protein within the environment of the live cell, allowing the detection and isolation of the target protein without the need for maintenance of the interaction after cell lysis. This technique is a valuable tool for studying biologically interesting small molecules with unknown mechanisms, both in the context of basic biology as well as drug discovery.

We describe here a method for identification of small molecule-binding proteins using photoaffinity labeling. The advantage of this technique is that binding and covalent labeling of the target proteins occurs within the live cellular environment, removing the risk of disrupting native protein structure and binding conditions upon cell lysis.

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several ...

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

Measuring Biomolecular DSC Profiles with Thermolabile Ligands to Rapidly Characterize Folding and Binding Interactions

Differential scanning calorimetry (DSC) is a powerful technique for quantifying thermodynamic parameters governing biomolecular folding and binding interactions. This information is critical in the design of new pharmaceutical compounds. However, many pharmaceutically relevant ligands are chemically unstable at the high temperatures used in DSC analyses. Thus, measuring binding interactions is challenging because the concentrations of ligands and thermally-converted products are constantly changing within the calorimeter cell. Here, we present a protocol using thermolabile ligands and DSC for rapidly obtaining thermodynamic and kinetic information on the folding, binding, and ligand conversion processes. We have applied our method to the DNA aptamer MN4 that binds to the thermolabile ligand cocaine. Using a new global fitting analysis that accounts for thermolabile ligand conversion, the complete set of folding and binding parameters are obtained from a pair of DSC experiments. In addition, we show that the rate constant for thermolabile ligand conversion may be obtained with only one supplementary DSC dataset. The guidelines for identifying and analyzing data from several more complicated scenarios are presented, including irreversible aggregation of the biomolecule, slow folding, slow binding, and rapid depletion of the thermolabile ligand.

We present a protocol for rapid characterization of biomolecular folding and binding interactions with thermolabile ligands using differential scanning calorimetry.

Differential scanning calorimetry (DSC) is a powerful technique for quantifying thermodynamic parameters governing biomolecular folding and binding ...

Nitrogen Cavitation and Differential Centrifugation Allows for Monitoring the Distribution of Peripheral Membrane Proteins in Cultured Cells

Cultured cells are useful for studying the subcellular distribution of proteins, including peripheral membrane proteins. Genetically encoded fluorescently tagged proteins have revolutionized the study of subcellular protein distribution. However, it is difficult to quantify the distribution with fluorescent microscopy, especially when proteins are partially cytosolic. Moreover, it is often important to study endogenous proteins. Biochemical assays such as immunoblots remain the gold standard for quantification of protein distribution after subcellular fractionation. Although there are commercial kits that aim to isolate cytosolic or certain membrane fractions, most of these kits are based on extraction with detergents, which may be unsuitable for studying peripheral membrane proteins that are easily extracted from membranes. Here we present a detergent-free protocol for cellular homogenization by nitrogen cavitation and subsequent separation of cytosolic and membrane-bound proteins by ultracentrifugation. We confirm the separation of subcellular organelles in soluble and pellet fractions across different cell types, and compare protein extraction among several common non-detergent-based mechanical homogenization methods. Among several advantages of nitrogen cavitation is the superior efficiency of cellular disruption with minimal physical and chemical damage to delicate organelles. Combined with ultracentrifugation, nitrogen cavitation is an excellent method to examine the shift of peripheral membrane proteins between cytosolic and membrane fractions.

Here we present protocols for detergent-free homogenization of cultured mammalian cells based on nitrogen cavitation and subsequent separation of cytosolic and membrane-bound proteins by ultracentrifugation. This method is ideal for monitoring the partitioning of peripheral membrane proteins between soluble and membrane fractions.

Cultured cells are useful for studying the subcellular distribution of proteins, including peripheral membrane proteins. Genetically encoded fluorescently ...

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 ...

Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe the application of this novel label-free technique to study the interaction of human EAG1 (hEAG1) channel proteins with the small molecule PIP2. hEAG1 channel has been recognized as potential therapeutic target because of its aberrant overexpression in cancers and a few gain-of-function mutations involved in some types of neurological diseases. We purified&#160;hEAG1 channel proteins from a mammalian stable expression system and measured the interaction with PIP2 by BLI. The successful measurement of the kinetics of binding between hEAG1 protein and PIP2 demonstrates that the BLI assay is a potential high-throughput approach used for novel small-molecule ligand screening in ion channel pharmacology.

The protocol here describes the interactions of purified hEAG1 ion channel protein with the small molecule lipid ligand phosphatidylinositol 4, 5-bisphosphate (PIP2). The measurement demonstrates that BLI could be a potential method for novel small-molecule ion channel ligand screening.

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe ...

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

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 ...

Analysis of AtHIRD11 Intrinsic Disorder and Binding Towards Metal Ions by Capillary Gel Electrophoresis and Affinity Capillary Electrophoresis

Plants are strongly dependent on their environment. In order to adjust to stressful changes (e.g., drought and high salinity), higher plants evolve classes of intrinsically disordered proteins (IDPs) to reduce oxidative and osmotic stress. This article uses a combination of capillary gel electrophoresis (CGE) and mobility shift affinity electrophoresis (ACE) in order to describe the binding behavior of different conformers of the IDP AtHIRD11 from Arabidopsis thaliana. CGE is used to confirm the purity of AtHIRD11 and to exclude fragments, posttranslational modifications, and other impurities as reasons for complex peak patterns. In this part of the experiment, the different sample components are separated by a viscous gel inside a capillary by their different masses and detected with a diode array detector. Afterward, the binding behavior of the sample towards various metal ions is investigated by ACE. In this case, the ligand is added to the buffer solution and the shift in migration time is measured in order to determine whether a binding event has occurred or not. One of the advantages of using the combination of CGE and ACE to determine the binding behavior of an IDP is the possibility to automate the gel electrophoresis and the binding assay. Furthermore, CGE shows a lower limit of detection than the classical gel electrophoresis and ACE is able to determine the manner of binding a ligand in a fast manner. In addition, ACE can also be applied to other charged species than metal ions. However, the use of this method for binding experiments is limited in its ability to determine the number of binding sites. Nevertheless, the combination of CGE and ACE can be adapted for characterizing the binding behavior of any protein sample towards numerous charged ligands.

This protocol combines the characterization of a protein sample by capillary gel electrophoresis and a fast-binding screening for charged ligands by affinity capillary electrophoresis. It is recommended for proteins with a flexible structure, such as intrinsically disordered proteins, to determine any differences in binding for different conformers.

Plants are strongly dependent on their environment. In order to adjust to stressful changes (e.g., drought and high salinity), higher plants evolve ...

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

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

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

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