TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol can be used to analyze phosphate uptake by KaiC proteins over time.</p>

[BEFORE_START]
<p>prepare gels for <a href="https://www.protocols.io/view/lowc-sds-page-gysbxwe" target="_blank">LowC SDS-PAGE</a></p>

[GUIDELINES]
<p><strong>references:</strong></p>
<p> </p>
<p><strong>Wiegard A, Dörrich AK, Deinzer HT, Beck C, Wilde A, Holtzendorff J, Axmann IM:</strong> Biochemical analysis of three putative KaiC clock proteins from <em>Synechocystis</em> sp. PCC 6803 suggests their functional divergence. <em>Microbiology 2013</em>, 159:948-958.</p>
<p> </p>
<p><strong>Axmann IM, Dühring U, Seeliger L, Arnold A, Vanselow JT, Kramer A, Wilde A:</strong> Biochemical evidence for a timing mechanism in <em>Prochlorococcus</em>. J Bacteriol 2009, 191:5342-5347</p>
<p> </p>
<p> </p>
<p><strong>required chemicals/buffers:</strong></p>
<p> </p>
<table border="1" width="604">
<tbody>
<tr>
<td width="124">
<p><strong>chemical/buffer</strong></p>
</td>
<td width="134">
<p> <strong>comments</strong></p>
</td>
</tr>
<tr>
<td width="124">
<p>[y-32P]ATP (3000 Ci/mmol)</p>
</td>
<td width="134">
<p>(3000 Ci/mmol), see materials tab</p>
</td>
</tr>
<tr>
<td width="124">
<p>reaction buffer (RB)</p>
</td>
<td width="134">
<p>depends on the protein of interest, see buffer table below</p>
</td>
</tr>
<tr>
<td width="124">
<p>5 x SDS Gel loading buffer</p>
</td>
<td width="134">
<p> see buffer table below</p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p><strong>buffer compositions:</strong></p>
<p> </p>
<table style="width: 540px;" border="1">
<tbody>
<tr>
<td style="width: 127px;"><strong>name</strong></td>
<td style="width: 165px;"><strong>ingredients</strong></td>
<td style="width: 266px;"><strong>reference</strong></td>
</tr>
<tr>
<td style="width: 127px;">5 x SDS Gel loading buffer</td>
<td style="width: 165px;">
<p>250 mM Tris-HCl (pH 6,8)</p>
<p>40 % (v/v) glycerol</p>
<p>8 % (w/v) SDS</p>
<p>100 mM DTT</p>
<p>0.1 % (w/v) bromophenol blue</p>
</td>
<td style="width: 266px;"><strong>Green MR and Sambrook J: </strong>Molecular cloning: a laboratory manual - 4th ed. <em>Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2012</em></td>
</tr>
<tr>
<td style="width: 127px;">reaction buffer for cyanobacterial Kai proteins</td>
<td style="width: 165px;">
<p>20 mM Tris/HCl (pH 8)</p>
<p>150 mM NaCl</p>
<p>0.5 mM EDTA</p>
<p>5 mM MgCl2</p>
<p>1 mM ATP</p>
</td>
<td style="width: 266px;"><strong>Nakajima M, Imai K, Ito H, Nishiwaki T, Murayama Y, Iwasaki H, Oyama T, Kondo T:</strong> Reconstitution of circadian oscillation of cyanobacterial KaiC phosphorylation in vitro. <em>Science 2005</em>, 308:414-415.</td>
</tr>
<tr>
<td style="width: 127px;">reaction buffer for cyanobacterial KaiC from <em>Thermococcus litoralis</em></td>
<td style="width: 165px;">
<p>50 mM HEPES (pH 7.2)</p>
<p>150 mM NaCl</p>
<p>5 mM MgCl2</p>
<p>1 mM ATP</p>
</td>
<td style="width: 266px;"> </td>
</tr>
<tr>
<td style="width: 127px;">reaction buffer for cyanobacterial KaiC from <em>Pycrococcus horikoshii</em></td>
<td style="width: 165px;">
<p>50 mM MES (pH 6)</p>
<p>150 mM NaCl</p>
<p>5 mM MgCl2</p>
<p>1 mM ATP</p>
</td>
<td style="width: 266px;"> </td>
</tr>
</tbody>
</table>
<p> </p>
<p><strong>required equipment:</strong></p>
<p> </p>
<table border="1" width="606">
<tbody>
<tr style="height: 16px;">
<td style="height: 16px;" width="202">
<p><strong>Device</strong></p>
</td>
</tr>
<tr style="height: 16px;">
<td style="height: 16px;" width="202">
<p>Heating block</p>
</td>
</tr>
<tr style="height: 16px;">
<td style="height: 16px;" width="202">
<p>Equipment for handling radioactive material</p>
</td>
</tr>
<tr style="height: 16px;">
<td style="height: 16px;" width="202">
<p>Gel dryer</p>
</td>
</tr>
<tr style="height: 16px;">
<td style="height: 16px;" width="202">
<p>radioactivity scanner (Fujifilm FLA-3000)</p>
</td>
</tr>
</tbody>
</table>

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