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[DESCRIPTION]
<p>SDS-PAGE using gels with low crosslinking of acrylamide and bisacrylamide. This protocol can be used to separate phosphorylation forms of KaiC proteins.</p>

[GUIDELINES]
<p><strong>references:</strong></p>
<p> </p>
<p><strong>1. SDS-PAGE</strong> modified from </p>
<p> </p>
<p><strong>Laemmli UK: </strong>Cleavage of structural proteins during the assembly of the head of bacteriophage 780 t4. <em>Nature 1970</em>, 227(5259):680–5,</p>
<p>and</p>
<p><strong>Green MR and Sambrook</strong> <strong>J:</strong> Molecular cloning: a laboratory manual - 4th ed. <em>Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2012</em></p>
<p> </p>
<p><strong>2. low C content</strong> according to</p>
<p><strong>Nishiwaki T, Satomi Y, Kitayama Y, Terauchi K, Kiyohara R, Takao T, Kondo T: </strong>A sequential program of dual phosphorylation of KaiC as a basis for circadian rhythm in cyanobacteria. <em>Embo J </em>2007, 26:4029-4037.</p>
<p> </p>
<p><strong>Possible modifícations:</strong></p>
<p> </p>
<p>The total acrylamide concentration (%T) and crosslinking of acrylamide and bis-acrylamide (%C) can be adjusted using the following formula (provided by Carl Roth <a href="https://www.carlroth.com/downloads/ba/en/3/BA_3037_EN.pdf" target="_blank">https://www.carlroth.com/downloads/ba/en/3/BA_3037_EN.pdf</a>)</p>
<p> </p>
<p>Vt = total gel volume (ml)</p>
<p>T = total gel concentration in % = % Acrylamide + % Bisacrylamide</p>
<p>C = % Crosslinking = (% Bisacrylamide x 100) / T</p>
<p>Va = Volume Gel A in ml</p>
<p>Vb = Volume Gel B in ml</p>
<p>Va = (T x (100 - C) x Vt ) / 3000</p>
<p>Vb = (T x C x Vt ) / 200</p>
<p> </p>
<p><strong>required chemicals/buffers</strong></p>
<p> </p>
<table style="width: 344px;">
<tbody>
<tr style="height: 20px;">
<td style="width: 417px; height: 20px;"><strong>chemical/buffer</strong></td>
<td style="width: 170px; height: 20px;"><strong>comments</strong></td>
</tr>
<tr style="height: 20px;">
<td style="width: 417px; height: 20px;">Rotiphorese® Gel A</td>
<td style="width: 170px; height: 20px;">see materials tab</td>
</tr>
<tr style="height: 20px;">
<td style="width: 417px; height: 20px;">Rotiphorese® Gel B</td>
<td style="width: 170px; height: 20px;">see materials tab</td>
</tr>
<tr style="height: 20px;">
<td style="width: 417px; height: 20px;">1.5 M Tris/HCl pH8.8</td>
<td style="width: 170px; height: 20px;"> </td>
</tr>
<tr style="height: 20px;">
<td style="width: 417px; height: 20px;">1 M Tris/HCl pH 6.8</td>
<td style="width: 170px; height: 20px;"> </td>
</tr>
<tr style="height: 20px;">
<td style="width: 417px; height: 20px;">TEMED</td>
<td style="width: 170px; height: 20px;"> </td>
</tr>
<tr style="height: 40px;">
<td style="width: 417px; height: 40px;">10 % (w/v) ammonium persulfate (APS)</td>
<td style="width: 170px; height: 40px;"> </td>
</tr>
<tr style="height: 20px;">
<td style="width: 417px; height: 20px;">20 % SDS (w/v)</td>
<td style="width: 170px; height: 20px;"> </td>
</tr>
<tr style="height: 20px;">
<td style="width: 417px; height: 20px;">2-propanol</td>
<td style="width: 170px; height: 20px;"> </td>
</tr>
<tr style="height: 20px;">
<td style="width: 417px; height: 20px;">1 x Tris-Glycine elctrophoresis buffer</td>
<td style="width: 170px; height: 20px;">see buffer table</td>
</tr>
<tr style="height: 20px;">
<td style="width: 417px; height: 20px;">5 x SDS Gel loading buffer</td>
<td style="width: 170px; height: 20px;">see buffer table</td>
</tr>
<tr style="height: 20px;">
<td style="width: 417px; height: 20px;">Rotiphorese® Blau R</td>
<td style="width: 170px; height: 20px;">see materials tab</td>
</tr>
<tr style="height: 20px;">
<td style="width: 417px; height: 20px;">7 % acetic acid</td>
<td style="width: 170px; height: 20px;"> </td>
</tr>
<tr style="height: 20px;">
<td style="width: 417px; height: 20px;">Destaining solution</td>
<td style="width: 170px; height: 20px;">see buffer table</td>
</tr>
</tbody>
</table>
<p> </p>
<p><strong>buffer compositions:</strong></p>
<p> </p>
<table style="width: 521px;">
<tbody>
<tr>
<td style="width: 241px;"><strong>name</strong></td>
<td style="width: 185px;"><strong>ingredients</strong></td>
<td style="width: 118px;"><strong>reference</strong></td>
</tr>
<tr>
<td style="width: 241px;">1 x Tris-Glycine elctrophoresis buffer</td>
<td style="width: 185px;">
<p>250 mM Tris</p>
<p>0.1 % (w/v) SDS</p>
<p>0.192 M Glycin</p>
</td>
<td style="width: 118px;">Green and Sambrook (2012)</td>
</tr>
<tr>
<td style="width: 241px;">5 x SDS Gel loading buffer</td>
<td style="width: 185px;">
<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: 118px;">Green and Sambrook (2012)</td>
</tr>
<tr>
<td style="width: 241px;">destaining solution</td>
<td style="width: 185px;">
<p>acetic acid: ethanol: H<sub>2</sub>O</p>
10:50:40 (v/v/v)</td>
<td style="width: 118px;"> </td>
</tr>
</tbody>
</table>
<p> </p>
<p><strong>required equipment:</strong></p>
<p> </p>
<table style="width: 337px;">
<tbody>
<tr>
<td style="width: 343px;">heating block</td>
</tr>
<tr>
<td style="width: 343px;">glass plates, spacers, combs, gel casting unit</td>
</tr>
<tr>
<td style="width: 343px;">gel chamber</td>
</tr>
<tr>
<td style="width: 343px;">syringe</td>
</tr>
<tr>
<td style="width: 343px;">power supply</td>
</tr>
<tr>
<td style="width: 343px;">benchtop centrifuge</td>
</tr>
<tr>
<td style="width: 343px;">horizontal shaker</td>
</tr>
</tbody>
</table>

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