We thank A. Riccio for helpful comments and to read the manuscript. This work was supported by the Medical Research Council (MRC) funding to the Cell Biology Unit and by a Human Frontier Science Program Grant (RGP0048/2009-C).

1. Enzymatic Reaction - day 1 (1 hour in the afternoon)
<ol>
	<li>The first step is to prepare 10-20 independent enzymatic reactions in which IP6K1 or VIP1 convert IP6 to the pyrophosphorylated isoforms.</li>
	<li>We use His-IP6K1 and GST-Vip1 enzymes purified from E. coli according to the protocol previously described17,18.</li>
	<li>Prepare 50 &mu;L reactions containing 1X reaction buffer (30 mM Hepes pH 6.8, 50 mM NaCl, 6 mM MgSO4, 1 mM DTT), 6 mM PhosphoCreatine (PCr), 25 U/mL CreatinePhosphoKinase (CPK), 5 mM ATP (Mg salt), 0.3 mM IP6, 0.05-0.1 &mu;g His-IP6K1 or GST-Vip1. Adjust volume with MiliQ ddH2O.</li>
	<li>Briefly spin the reaction and incubate at 37&deg;C overnight with rotation.</li>
</ol>
2. Polyacrylamide gel casting and loading - day 2 (4 hours in the afternoon)
<ol>
	<li>The polyacrylamide gel is prepared using 24 cm long,18 cm wide glass plates and 1.5 mm wide spacers. Usually a 16 lane or a preparative single lane comb is used.</li>
	<li>Prepare a mix (50 mL/gel) containing the following: 35.5% (w/v) Acrylamide:Bis-Acrylamide 19:1, 1X Tris/Borate/EDTA (TBE), 0.05% (w/v) ammonium persulfate (APS), 0.05% (w/v) Temed. Pour the mix between the pre-casted glass plates, insert the comb and let polymerize for 30-60 minutes at RT.</li>
	<li>Once the gel has polymerized, transfer the apparatus to the cold room and pre-run in 1X TBE for about 30-60 minutes at 200-300 Volts.</li>
	<li>Add 1X of OrangeG dye (10 mM Tris-HCl pH7.0, 1 mM EDTA, 30% glycerol, 0.1% OrangeG) to each reaction. Prepare a sample containing 2 nmol of IP6 to load as a standard control.</li>
	<li>Wash each well thoroughly with running buffer using a syringe and a 21G needle to remove any precipitate, then load the gel. Avoid loading on the side wells.</li>
	<li>Run the gel overnight at 450-550 Volts (7 mAmp/gel), until the OrangeG dye band is within the last 10 cm from the bottom of the gel.</li>
</ol>
3. Isolation of IP7 - day 3 (4 hours) and day 4 (6-7 hour SpeedVac drying process)
<ol>
	<li>Disassemble the gel apparatus and carefully remove one glass plate leaving the gel on the other one. Cut a small portion of the gel from just above the OrangeG dye band to the bottom containing the IP6 standard and one sample lane, as shown in Figure 1.</li>
	<li>Stain the cut portion of the gel with Toluidine Blue (0.1% (w/v) toluidine blue, 20% (w/v) methanol, 2% (w/v) glycerol) for a few minutes (1-3 min) or until the inositol pyrophosphate band appears. Put the glass plate previously removed back on top of the gel to prevent the unstained gel from drying.</li>
</ol>
The IP7 band should be visible since it runs slightly slower than the IP6 standard. ATP, which runs faster than IP6, should also be visible (Figure 1). Transfer the stained portion of the gel in a de-staining solution (20% (w/v) methanol) for a few minutes, wash away any excess of Toluidine Blue and reposition the gel with the unstained gel.
If visualization of higher pyrophosphorylated inositol isoforms (IP8 and IP9) is required, stain the gel with Toluidine Blue staining solution for 20 minutes at room temperature. Subsequently, wash away the Toluidine Blue with the de-staining solution for about 15 minutes.
<ol start="3">
	<li>With a razor blade cut the IP7 band on the unstained portion of the gel using as reference the IP7 migrating position determined with the stained gel (Figure 1).</li>
	<li>Put the IP7 band that was cut from the gel on a 15 mL tube and add 10 mL of MilliQ ddH2O. Put tubes in rotation for 10 minutes at room temperature. Discard the liquid to remove excess of TBE and microscopic acrylamide particles.</li>
	<li>Subsequently, perform two dehydration-hydration cycles. Add 5 mL of 50% (w/v) methanol to the tube with the gel containing IP7 and rotate at room temperature for 2 hours. Transfer the gel slice to a new 15 mL tube containing 5 mL of MilliQ ddH2O and rotate at room temperature for 2 hours. Do not discard the methanol and MilliQ H2O from the tubes. Repeat the dehydration-hydration cycle once more by re-transferring the polyacrylamide gel in the 15 mL tubes previously used. One of the washes can be performed over night.</li>
	<li>To concentrate the eluted IP7, dry the 10 mL together (5 mL MilliQddH2O and 5 mL 50% (w/v) methanol) using a SpeedVac heated at 60&deg;C.</li>
	<li>Once the samples are nearly dry, transfer the remaining liquid (300-600 &mu;l) to a1.5 mL centrifuge tube and spin for 2 minutes at 5000 rpm.</li>
	<li>Collect supernatant and transfer into a fresh 1.5 mL centrifuge tube; leave the bottom 20-30 &mu;L since it may contain acrylamide particles.</li>
	<li>If necessary continue the drying process using an unheated SpeedVac. The recovery of IP7 is dramatically reduced if the samples dry completely, therefore terminate the drying process when the samples reach the volume of 100-300 &mu;L.</li>
</ol>
4. Determination of IP7 concentration and purity.
<ol>
	<li>Use 2-5 &mu;L of the recovered IP7 sample to run on a PAGE gel, similarly to Sections 2.2-2.5. Load several dilutions of IP6 (i.e. 0.5, 1, 2, 4 nmol) as concentration standard and 4 nmol of Poly-P marker. After running the gel, visualize the inositol pyrophosphate isoforms by staining and de-staining the entire gel with Toluidine Blue solution, following the procedure described in Section 3.2 (Figure 2A).</li>
	<li>After Toluidine staining, the concentrations can be determined by scanning the gel and comparing the differences in intensity between IP6 and IP7, using imaging software such as Image-J, as shown in Figure 2B.</li>
</ol>
5. Representative Results:
The preparative enzymatic conversion of IP6 to IP7 using IP6K1 and VIP1 enzymes can be easily resolved using PAGE analysis (Figure 1). The loading of IP6 as a size control together with Toluidine Blue gel staining allows the identification of the pyrophosphorylated derivates, since they run slower depending on the number of phosphate groups present on the inositol ring. The procedure described above allows the easy purification of IP7. The analysis of the purified inositol pyrophosphate by PAGE revealed the purity of our IP7 (Figure 2A). Interestingly, the 1/3PP-IP5 isomer of IP7 product of VIP1 migrates slightly slower than the 5PP-IP5 isomer of IP7 that is generated by the IP6K1. Use of IP6 standards permit an easy quantification of the concentration of the purified IP7 (Figure 2B). Before using IP7 for further experiments, its biological activity can be assessed (Figure 3). 5PP-IP5 is incubated with VIP1 and with the IP7 phosphatase DDP1 (diphosphoinositol polyphosphate phosphohydrolase). Routinely, the purified IP7 is converted to IP8 by VIP1 and to IP6 by DDP1 (Figure 3).
<img alt="Figure 1" src="/files/ftp_upload/3027/3027fig1.jpg" /> 
Figure 1: Toluidine staining of PAGE and isolation of the IP7 band. The portion of the gel containing the standard (IP6) was cut and stained using a Toluidine Blue solution. The three bands represent (top to bottom) IP7, IP6 and ATP. The stained portion of the gel was then aligned with the remaining of the gel. This allows the localization of the portion of the gel containing IP7, which can then be cut and purified (dashed box).
<img alt="Figure 2" src="/files/ftp_upload/3027/3027fig2.jpg" /> 
Figure 2: PAGE analysis of IP6K1 and VIP1 reaction products. A) Analysis of IP6 (4, 2, 1, 0.5 nmol) by Toluidine Blue staining was used in order to determine the IP7 concentration purified from both IP6K1 (5PP-IP5) and VIP1 (1/3PP-IP5) reactions. B) Scatter plot analysis to determine the concentration of the purified IP7. Concentrations were determined according to band intensity, calculated using imageJ software, compared to pre-determined amounts of IP6. The X-axis represents intensities; the Y-axis represents concentrations expressed in nmol.
<img alt="Figure 3" src="/files/ftp_upload/3027/3027fig3.jpg" /> 
Figure 3: Analysis of IP7 biological activity. To determine the quality of the purified IP7 we incubated 5PP-IP5 (IP6K1 generated IP7) with VIP1 or with the IP7 phosphatase DDP1 and then resolved the reaction on PAGE. The DAPI and Toluidine staining revealed the expected production of IP8 by VIP1 and the conversion of IP7 to IP6 by DDP1.

<ol>
	<li>Bennett, M., Onnebo, S. M., Azevedo, C., Saiardi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+pyrophosphates:+metabolism+and+signaling.">Inositol pyrophosphates: metabolism and signaling.</a> Cell Mol Life Sci. 63, 552-564 (2006).</li><li>Burton, A., Hu, X., Saiardi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+inositol+pyrophosphates+signalling+molecules?.">Are inositol pyrophosphates signalling molecules?.</a> J Cell Physiol. 220, 8-15 (2009).</li><li>Barker, C. J., Illies, C., Gaboardi, G. C., Berggren, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+pyrophosphates:+structure,+enzymology+and+function.">Inositol pyrophosphates: structure, enzymology and function.</a> Cell Mol Life Sci. 66, 3851-3871 (2009).</li><li>Shears, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diphosphoinositol+polyphosphates:+metabolic+messengers.">Diphosphoinositol polyphosphates: metabolic messengers.</a> Mol Pharmacol. 76, 236-252 (2009).</li><li>Saiardi, A., Erdjument-Bromage, H., Snowman, A. M., Tempst, P., Snyder, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+diphosphoinositol+pentakisphosphate+by+a+newly+identified+family+of+higher+inositol+polyphosphate+kinases.">Synthesis of diphosphoinositol pentakisphosphate by a newly identified family of higher inositol polyphosphate kinases.</a> Curr Biol. 9, 1323-1326 (1999).</li><li>Draskovic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+hexakisphosphate+kinase+products+contain+diphosphate+and+triphosphate+groups.">Inositol hexakisphosphate kinase products contain diphosphate and triphosphate groups.</a> Chem Biol. 15, 274-286 (2008).</li><li>Mulugu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conserved+family+of+enzymes+that+phosphorylate+inositol+hexakisphosphate.">A conserved family of enzymes that phosphorylate inositol hexakisphosphate.</a> Science. 316, 106-109 (2007).</li><li>Fridy, P. C., Otto, J. C., Dollins, D. E., York, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+characterization+of+two+human+VIP1-like+inositol+hexakisphosphate+and+diphosphoinositol+pentakisphosphate+kinases.">Cloning and characterization of two human VIP1-like inositol hexakisphosphate and diphosphoinositol pentakisphosphate kinases.</a> J Biol Chem. 282, 30754-30762 (2007).</li><li>Illies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+of+inositol+pyrophosphates+for+full+exocytotic+capacity+in+pancreatic+beta+cells.">Requirement of inositol pyrophosphates for full exocytotic capacity in pancreatic beta cells.</a> Science. 318, 1299-1302 (2007).</li><li>Saiardi, A., Resnick, A. C., Snowman, A. M., Wendland, B., Snyder, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+pyrophosphates+regulate+cell+death+and+telomere+length+through+phosphoinositide+3-kinase-related+protein+kinases.">Inositol pyrophosphates regulate cell death and telomere length through phosphoinositide 3-kinase-related protein kinases.</a> Proc Natl Acad Sci U S A. 102, 1911-1914 (2005).</li><li>York, S. J., Armbruster, B. N., Greenwell, P., Petes, T. D., York, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+diphosphate+signaling+regulates+telomere+length.">Inositol diphosphate signaling regulates telomere length.</a> J Biol Chem. 280, 4264-4269 (2005).</li><li>Luo, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+pyrophosphates+mediate+chemotaxis+in+Dictyostelium+via+pleckstrin+homology+domain-PtdIns(3,4,5)P3+interactions.">Inositol pyrophosphates mediate chemotaxis in Dictyostelium via pleckstrin homology domain-PtdIns(3,4,5)P3 interactions.</a> Cell. 114, 559-572 (2003).</li><li>Saiardi, A., Sciambi, C., McCaffery, J. M., Wendland, B., Snyder, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+pyrophosphates+regulate+endocytic+trafficking.">Inositol pyrophosphates regulate endocytic trafficking.</a> Proc Natl Acad Sci U S A. 99, 14206-14211 (2002).</li><li>Auesukaree, C., Tochio, H., Shirakawa, M., Kaneko, Y., Harashima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plc1p,+Arg82p,+and+Kcs1p,+enzymes+involved+in+inositol+pyrophosphate+synthesis,+are+essential+for+phosphate+regulation+and+polyphosphate+accumulation+in+Saccharomyces+cerevisiae.">Plc1p, Arg82p, and Kcs1p, enzymes involved in inositol pyrophosphate synthesis, are essential for phosphate regulation and polyphosphate accumulation in Saccharomyces cerevisiae.</a> J Biol Chem. 280, 25127-25133 (2005).</li><li>Azevedo, C., Burton, A., Ruiz-Mateos, E., Marsh, M., Saiardi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+pyrophosphate+mediated+pyrophosphorylation+of+AP3B1+regulates+HIV-1+Gag+release.">Inositol pyrophosphate mediated pyrophosphorylation of AP3B1 regulates HIV-1 Gag release.</a> Proc Natl Acad Sci U S A. 106, 21161-21166 (2009).</li><li>Bhandari, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+pyrophosphorylation+by+inositol+pyrophosphates+is+a+posttranslational+event.">Protein pyrophosphorylation by inositol pyrophosphates is a posttranslational event.</a> Proc Natl Acad Sci U S A. 104, 15305-15310 (2007).</li><li>Azevedo, C., Burton, A., Bennett, M., Onnebo, S. M., Saiardi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+InsP7+by+the+Inositol+Hexakisphosphate+Kinase+1+(IP6K1).">Synthesis of InsP7 by the Inositol Hexakisphosphate Kinase 1 (IP6K1).</a> Methods Mol Biol. 645, 73-85 (2010).</li><li>Otto, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+analysis+of+inositol+phosphate+kinases.">Biochemical analysis of inositol phosphate kinases.</a> Methods Enzymol. 434, 171-185 (2007).</li><li>Losito, O., Szijgyarto, Z., Resnick, A. C., Saiardi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+pyrophosphates+and+their+unique+metabolic+complexity:+analysis+by+gel+electrophoresis.">Inositol pyrophosphates and their unique metabolic complexity: analysis by gel electrophoresis.</a> PLoS One. 4, e5580-e5580 (2009).</li></ol>

No conflicts of interest declared.

The use of inositol pyrophosphate in biochemistry is severely limited by the commercial unavailability of such compounds and the poor sensitivity of the existing detection methods. The combination of PAGE, which enables the separation of molecules possessing different number of phosphate groups, and Toluidine Blue (Figure 1), a metachromatic dye which binds to phosphate groups, enables the easy detection of inositol pyrophoshate isoforms opening new avenues of research20.
The described use of PAGE technology to purify inositol pyrophosphate products of the enzymatic reaction carried outby either IP6K1 or VIP1 is a simple, economic and reliable method that allows for the production of large amounts of high quality IP7. The method described above is not limited to the simple purification of IP7 but minor modifications of the described protocol may allow the purification of a different range of inositol pyrophosphates. Higher phosphorylated inositol pyrophosphate isoforms, containing more than eight phosphate groups can be detected using IP7 or different amounts of IP6 as a substrate20,6. These inositol pyrophosphates can be detected by increasing the length of the staining procedure and subsequently purified (section 3.2). Moreover, the use of IP5 as substrate for the enzymatic reaction would allow the purification of PP-IP5 and other inositol pyrophosphates containing a hydroxyl group on the inositol ring.
In conclusion, this undemanding method allows for the reliable purification of milligram quantities of inositol pyrophosphates with widely available instruments, thus opening new avenues for this exciting research field.

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
		</tr>
		<tr>
			<td>Phytic Acid (IP6)</td>
			<td>Sigma-Aldrich</td>
			<td>P8810</td>
		</tr>
		<tr>
			<td>Poly-P (sodium hexametaphosphate)</td>
			<td>Sigma-Aldrich</td>
			<td>P8510</td>
		</tr>
		<tr>
			<td>ATP-Mg2+ salt</td>
			<td>Sigma-Aldrich</td>
			<td>A9187</td>
		</tr>
		<tr>
			<td>OrangeG</td>
			<td>Sigma-Aldrich</td>
			<td>O3756</td>
		</tr>
		<tr>
			<td>PhosphoCreatine (PCr)</td>
			<td>Sigma-Aldrich</td>
			<td>P7936</td>
		</tr>
		<tr>
			<td>CreatinePhospho Kinase (CPK)</td>
			<td>Sigma-Aldrich</td>
			<td>C3755</td>
		</tr>
		<tr>
			<td>GST-Vip1</td>
			<td>17</td>
			<td>17</td>
		</tr>
		<tr>
			<td>His-IP6K1</td>
			<td>18</td>
			<td>18</td>
		</tr>
		<tr>
			<td>His-Ddp1</td>
			<td>Available in lab</td>
			<td>Available in lab</td>
		</tr>
		<tr>
			<td>Acrylamide:Bis-Acrylamide 19:1 (40%)</td>
			<td>Flowgen</td>
			<td>H16972</td>
		</tr>
		<tr>
			<td>Ammonium Persulfate (APS)</td>
			<td>Sigma-Aldrich</td>
			<td>A9164</td>
		</tr>
		<tr>
			<td>Tris/Borate/EDTA (TBE)</td>
			<td>Sigma-Aldrich</td>
			<td>T 9060</td>
		</tr>
		<tr>
			<td>Temed</td>
			<td>BDH</td>
			<td>43083G</td>
		</tr>
		<tr>
			<td>Toluidine Blue</td>
			<td>Sigma-Aldrich</td>
			<td>198161</td>
		</tr>
		<tr>
			<td>SpeedVac</td>
			<td>Christ</td>
			<td>100218</td>
		</tr>
		<tr>
			<td>Gel apparatus</td>
			<td>Hoefer</td>
			<td>SE600</td>
		</tr>
		<tr>
			<td>Vacuum manifold</td>
			<td>Christ</td>
			<td>Alpha 2-4</td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>ABM Greiffenberger</td>
			<td>4EKF63CX</td>
		</tr>		
	</tbody>
</table>

preparation of quality inositol pyrophosphates

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells are inositol pentakisphosphate (IP5) and inositol hexakisphosphate (phytic acid or IP6). IP5 and IP6 are the precursors of inositol pyrophosphate molecules that contain one or more pyrophosphate bonds1. Phosphorylation of IP6 generates diphoshoinositolpentakisphosphate (IP7 or PP-IP5) and bisdiphoshoinositoltetrakisphosphate (IP8 or (PP)2-IP4). Inositol pyrophosphates have been isolated from all eukaryotic organisms so far studied. In addition, the two distinct classes of enzymes responsible for inositol pyrophosphate synthesis are highly conserved throughout evolution2-4.
The IP6 kinases (IP6Ks) posses an enormous catalytic flexibility, converting IP5 and IP6 to PP-IP4 and IP7 respectively and subsequently, by using these products as substrates, promote the generation of more complex molecules5,6. Recently, a second class of pyrophosphate generating enzymes was identified in the form of the yeast protein VIP1 (also referred as PP-IP5K), which is able to convert IP6 to IP7 and IP87,8.
Inositol pyrophosphates regulate many disparate cellular processes such as insulin secretion9, telomere length10,11, chemotaxis12, vesicular trafficking13, phosphate homeostasis14 and HIV-1 gag release15. Two mechanisms of actions have been proposed for this class of molecules. They can affect cellular function by allosterically interacting with specific proteins like AKT16. Alternatively, the pyrophosphate group can donate a phosphate to pre-phosphorylated proteins17. The enormous potential of this research field is hampered by the absence of a commercial source of inositol pyrophosphates, which is preventing many scientists from studying these molecules and this new post-translational modification. The methods currently available to isolate inositol pyrophosphates require sophisticated chromatographic apparatus18,19. These procedures use acidic conditions that might lead to inositol pyrophosphate degradation20 and thus to poor recovery. Furthermore, the cumbersome post-column desalting procedures restrict their use to specialized laboratories.
In this study we describe an undemanding method for the generation, isolation and purification of the products of the IP6-kinase and PP-IP5-kinases reactions. This method was possible by the ability of polyacrylamide gel electrophoresis (PAGE) to resolve highly phosphorylated inositol polyphosphates20. Following IP6K1 and PP-IP5K enzymatic reactions using IP6 as the substrate, PAGE was used to separate the generated inositol pyrophosphates that were subsequently eluted in water.

Watch this Scientific Journal Video about Preparation of Quality Inositol Pyrophosphates at JoVE.com

Preparation of Quality Inositol Pyrophosphates

Inositol pyrophosphates play an important role in human pathologies such cancer, diabetes and obesity; however, the exact mechanism of action is a matter of dispute. The lack of commercially available inositol pyrophosphates renders detailed studies problematic. Here we describe a simple protocol to produce and isolate milligrams of inositol pyrophosphates.

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells ...

preparation-of-quality-inositol-pyrophosphates

Research

JoVE Journal

Biology

13.0K Views.  University College London. Inositol pyrophosphates play an important role in human pathologies such cancer, diabetes and obesity; however, the exact mechanism of action is a matter of dispute. The lack of commercially available inositol pyrophosphates renders detailed studies problematic. Here we describe a simple protocol to produce and isolate milligrams of inositol pyrophosphates.

Video: Preparation of Quality Inositol Pyrophosphates

Horizontal Slice Preparation of the Retina

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of retinal neurons, such as amacrine cells, expand their dendrites horizontally, so that the morphology of the cells is supposed to be severely damaged in the vertical slices. In the whole-mount preparation, especially for patch-clamp recordings, retinal neurons in the middle layer are not easily accessible due to the extensive coverage of glial cell (Mueller cell) s endfeets. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact. The slice was made horizontally at the inner layer of the retina using a vibratome slicer after the retina was embedded in the low-temperature melting agarose gel. In this horizontal slice preparation of the retina, we studied the function of retinal neurons compared with their morphology, by using patch-clamp recording, calcium imaging technique, immunocytochemistry, and single-cell RT-PCR.

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact.

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of ...

DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, different physiological or pathological states, response to therapy, and, in general, any condition that is being experimentally tested1. Expression profiling of neural tissues requires isolation of high quality RNA, amplification of the isolated RNA and hybridization to DNA microarrays. In this article we describe protocols for reproducible microarray experiments from brain tumor tissue2. We will start by performing a quality control analysis of isolated RNA samples with Agilent's 2100 Bioanalyzer "lab-on-a-chip" technology. High quality RNA samples are critical for the success of any microarray experiment, and the 2100 Bioanalyzer provides a quick, quantitative measurement of the sample quality. RNA samples are then amplified and labeled by performing reverse transcription to obtain cDNA, followed by in vitro transcription in the presence of labeled nucleotides to produce labeled cRNA. By using a dual-color labeling kit, we will label our experimental sample with Cy3 and a reference sample with Cy5. Both samples will then be combined and hybridized to Agilent's 4x44 K arrays. Dual-color arrays offer the advantage of a direct comparison between two RNA samples, thereby increasing the accuracy of the measurements, in particular for small changes in expression levels, because the two RNA samples are hybridized competitively to a single microarray. The arrays will be scanned at the two corresponding wavelengths, and the ratio of Cy3 to Cy5 signal for each feature will be used as a direct measurement of the relative abundance of the corresponding mRNA. This analysis identifies genes that are differentially expressed in response to the experimental conditions being tested.

We demonstrate the use of DNA microarrays for expression profiling of the nervous system. We describe RNA quality control, sample labeling, and array hybridization and scanning.

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, ...

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, the effect of tissue elasticity on cell function is a major area of mechanobiology research because tissue stiffness modulates with disease, development, and injury. Static tissue-mimicking materials, or materials that cannot alter stiffness once cells are plated, are predominately used to investigate the effects of tissue stiffness on cell functions. While information gathered from static studies is valuable, these studies are not indicative of the dynamic nature of the cellular microenvironment in vivo. To better address the effects of dynamic stiffness on cell function, we developed a DNA-crosslinked polyacrylamide hydrogel system (DNA gels). Unlike other dynamic substrates, DNA gels have the ability to decrease or increase in stiffness after fabrication without stimuli. DNA gels consist of DNA crosslinks that are polymerized into a polyacrylamide backbone. Adding and removing crosslinks via delivery of single-stranded DNA allows temporal, spatial, and reversible control of gel elasticity. We have shown in previous reports that dynamic modulation of DNA gel elasticity influences fibroblast and neuron behavior. In this report and video, we provide a schematic that describes the DNA gel crosslinking mechanisms and step-by-step instructions on the preparation DNA gels.

Our laboratory has developed DNA-crosslinked polyacrylamide hydrogels, a dynamic hydrogel system, to better understand the effects of modulating tissue stiffness on cell function. Here, we provide schematics, descriptions, and protocols to prepare these hydrogels.

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, ...

Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state by transmission electron microscopy (TEM). This can be achieved with very low quantity of protein and in the buffer of choice, without the use of any stain, which is very useful to determine structure-function correlations of macromolecules. When combined with single-particle image processing, the technique has found widespread usefulness for 3D structural determination of purified macromolecules. 
The protocol presented here explains how to perform cryoEM and examines the causes of most commonly encountered problems for rational troubleshooting; following all these steps should lead to acquisition of high quality cryoEM images. The technique requires access to the electron microscope instrument and to a vitrification device. Knowledge of the 3D reconstruction concepts and software is also needed for computerized image processing. Importantly, high quality results depend on finding the right purification conditions leading to a uniform population of structurally intact macromolecules. 
The ability of cryoEM to visualize macromolecules combined with the versatility of single particle image processing has proven very successful for structural determination of large proteins and macromolecular machines in their near-native state, identification of their multiple components by 3D difference mapping, and creation of pseudo-atomic structures by docking of x-ray structures. The relentless development of cryoEM instrumentation and image processing techniques for the last 30 years has resulted in the possibility to generate de novo 3D reconstructions at atomic resolution level.

This paper describes the cryo-electron microscopy methodology, which is used to obtain high quality microscopic images of macromolecules in their near-native state. The method yields images suitable for further computerized processing using the single particle approach, devised to generate the 3D reconstruction of a macromolecule.

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state ...

A Web Tool for Generating High Quality Machine-readable Biological Pathways

PathWhiz is a web server built to facilitate the creation of colorful, interactive, visually pleasing pathway diagrams that are rich in biological information. The pathways generated by this online application are machine-readable and fully compatible with essentially all web-browsers and computer operating systems. It uses a specially developed, web-enabled pathway drawing interface that permits the selection and placement of different combinations of pre-drawn biological or biochemical entities to depict reactions, interactions, transport processes and binding events. This palette of entities consists of chemical compounds, proteins, nucleic acids, cellular membranes, subcellular structures, tissues, and organs. All of the visual elements in it can be interactively adjusted and customized. Furthermore, because this tool is a web server, all pathways and pathway elements are publicly accessible. This kind of pathway &#34;crowd sourcing&#34; means that PathWhiz already contains a large and rapidly growing collection of previously drawn pathways and pathway elements. Here we describe a protocol for the quick and easy creation of new pathways and the alteration of existing pathways. To further facilitate pathway editing and creation, the tool contains replication and propagation functions. The replication function allows existing pathways to be used as templates to create or edit new pathways. The propagation function allows one to take an existing pathway and automatically propagate it across different species. Pathways created with this tool can be &#34;re-styled&#34; into different formats (KEGG-like or text-book like), colored with different backgrounds, exported to BioPAX, SBGN-ML, SBML, or PWML data exchange formats, and downloaded as PNG or SVG images. The pathways can easily be incorporated into online databases, integrated into presentations, posters or publications, or used exclusively for online visualization and exploration. This protocol has been successfully applied to generate over 2,000 pathway diagrams, which are now found in many online databases including HMDB, DrugBank, SMPDB, and ECMDB.

PathWhiz is a comprehensive, online pathway drawing tool for generating biochemical and biological pathways. It uses publicly accessible databases and easily expandable palettes consisting of pre-drawn pathway components. This protocol describes how to easily build new pathways, replicate and edit existing pathways, and propagate previously drawn pathways to different organisms.

PathWhiz is a web server built to facilitate the creation of colorful, interactive, visually pleasing pathway diagrams that are rich in biological ...

Sperm Collection of Differential Quality Using Density Gradient Centrifugation

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with fertilizing ability are required to interact with a female gamete to ensure fertilization. As such, the fertilizing ability of individual sperm cells is critical for successful reproduction. Density gradient centrifugation has been utilized for several decades as a reproducible, fast, efficient, effective and extremely adaptable method to collect only high-quality sperm to be used in assisted reproductive technology. The protocols we described herein focus on the utilization of the discontinuous Percoll density gradient centrifugation (PDGC) technique to isolate three distinct populations of rooster sperm by their quality. We were able to collect low-, medium- and high-quality sperm. We also describe reproducible protocols that entail determining fertility potential of sperm by assessing their viability, mobility and penetrability. Collection of sperm by their quality using PDGC technique would be useful to accurately and thoroughly characterize sperm with differential fertility potential.

In this paper, we aim to describe the performance of the density gradient centrifugation technique and its application in sperm physiology research.

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with ...

Field Identification of Matricaria chamomilla using a Portable qPCR System

Quality control in botanical products begins with the raw material supply. Traditionally, botanical identification is performed through morphological assessment and chemical analytical methods. However, the lack of availability of botanists, especially in recent years, coupled with the need to enhance quality control to combat the stresses on the supply chain brought by increasing consumer demand and climate change, necessitates alternative approaches. The goal of this protocol is to facilitate botanical species identification using a portable qPCR system on the field or in any setting, where access to laboratory equipment and expertise is limited. Target DNA is amplified using dye-based qPCR, with DNA extracted from botanical reference materials serving as a positive control. The target DNA is identified by its specific amplification and matching its melting peak against the positive control. A detailed description of the steps and parameters, from hands-on field sample collection, to DNA extraction, PCR amplification, followed by data interpretation, has been included to ensure that readers can replicate this protocol. The results produced align with traditional laboratory botanical identification methods. The protocol is easy to perform and cost-effective, enabling quality testing on raw materials as close to the point of origin of the supply chain as possible.

Field Identification of Matricaria chamomilla using a Portable qPCR System

Presented here is a protocol for field identification of Matricaria chamomilla using a portable qPCR system. This easy-to-perform protocol is ideal as a method to confirm the identity of a botanical species at locations where access to laboratory equipment and expertise is limited, such as farms and warehouses.

Quality control in botanical products begins with the raw material supply. Traditionally, botanical identification is performed through morphological ...