1. Work at room temp- make cell suspension at concentration of 1-2x106 cells/ml of complete medium (with FCS) in a falcon 15ml if 10mls.
2. Vortex cell suspension at medium speed and add drop by drop 1:10 of 10x fixation buffer.
3. Incubate for 10 minutes at room temp on a rotating platform (time of incubation has to be optimized depending on the protein of interest).
4. To stop the fixation reaction, while vortexing at medium speed, add 1:20 of 20x glycine buffer, invert tubes twice and incubate on ICE for at least 5 minutes. From now on work on ICE.
5. Spin at 1000xg for 4 min at 4°C (~2100 rpm in buckets).
6. Discard the supernatant and resuspend with at least 5 mls of ice cold PBS. Incubate on ice for 2 minutes.
7. Spin at 1250xg (~2300rpm) for 4 min at 4°C.
8. Discard the supernatant.
9. Carefully resuspend the pellet with 1ml of ice cold PBS and transfer to 1.5ml eppendorf.
10. Spin 4 min at 1250xg.
11. Discard as much as you can of the supernatant (aspirate) without affecting the cell pellet.
12. Snap freeze the pellet in liquid nitrogen (if you wish to stop here), or continue to cell lysis and sonication.
Category:
Materials:
<p>* 20x glycine buffer: 9.38g for 50ml water (dissolve at 42°C)</p>
<p>* Ice cold PBS</p>
<p>* 10X fixation buffer (prepare fresh or use it within 2 weeks):</p>
<table class="MsoTableGrid" style="border-collapse: collapse; width: 20px;" border="1" cellpadding="0" cellspacing="0">
<tbody>
<tr style="mso-yfti-irow: 0; mso-yfti-firstrow: yes;">
<td style="width: 110.7pt; border: 1.0pt; mso-border-themecolor: text1; mso-border-alt: .5pt; background: #A6A6A6; mso-background-themecolor: background1; mso-background-themeshade: 166; padding: 0in 5.4pt 0in 5.4pt;" valign="top" width="111">
<p class="MsoNormal">Component</p>
</td>
<td style="width: 110.7pt; border: 1.0pt; mso-border-themecolor: text1; border-left: none; mso-border-left-alt: .5pt; mso-border-left-themecolor: text1; mso-border-alt: .5pt; background: #A6A6A6; mso-background-themecolor: background1; mso-background-themeshade: 166; padding: 0in 5.4pt 0in 5.4pt;" valign="top" width="111">
<p class="MsoNormal">Stock conc</p>
</td>
<td style="width: 110.7pt; border: 1.0pt; mso-border-themecolor: text1; border-left: none; mso-border-left-alt: .5pt; mso-border-left-themecolor: text1; mso-border-alt: .5pt; background: #A6A6A6; mso-background-themecolor: background1; mso-background-themeshade: 166; padding: 0in 5.4pt 0in 5.4pt;" valign="top" width="111">
<p class="MsoNormal">Final conc 10x (1x)</p>
</td>
<td style="width: 110.7pt; border: 1.0pt; mso-border-themecolor: text1; border-left: none; mso-border-left-alt: .5pt; mso-border-left-themecolor: text1; mso-border-alt: .5pt; background: #A6A6A6; mso-background-themecolor: background1; mso-background-themeshade: 166; padding: 0in 5.4pt 0in 5.4pt;" valign="top" width="111">
<p class="MsoNormal">Volume for 10 mls</p>
</td>
</tr>
<tr style="mso-yfti-irow: 1;">
<td style="width: 110.7pt; border: 1.0pt; mso-border-themecolor: text1; border-top: none; mso-border-top-alt: .5pt; mso-border-top-themecolor: text1; mso-border-alt: .5pt; padding: 0in 5.4pt 0in 5.4pt;" valign="top" width="111">
<p class="MsoNormal">Formaldehyde</p>
</td>
<td style="width: 110.7pt; border-top: none; border-left: none; border-bottom: 1.0pt; mso-border-bottom-themecolor: text1; border-right: 1.0pt; mso-border-right-themecolor: text1; mso-border-top-alt: .5pt; mso-border-top-themecolor: text1; mso-border-left-alt: .5pt; mso-border-left-themecolor: text1; mso-border-alt: .5pt; mso-border-themecolor: text1; padding: 0in 5.4pt 0in 5.4pt;" valign="top" width="111">
<p class="MsoNormal">37%</p>
</td>
<td style="width: 110.7pt; border-top: none; border-left: none; border-bottom: 1.0pt; mso-border-bottom-themecolor: text1; border-right: 1.0pt; mso-border-right-themecolor: text1; mso-border-top-alt: .5pt; mso-border-top-themecolor: text1; mso-border-left-alt: .5pt; mso-border-left-themecolor: text1; mso-border-alt: .5pt; mso-border-themecolor: text1; padding: 0in 5.4pt 0in 5.4pt;" valign="top" width="111">
<p class="MsoNormal">11% (1%)</p>
</td>
<td style="width: 110.7pt; border-top: none; border-left: none; border-bottom: 1.0pt; mso-border-bottom-themecolor: text1; border-right: 1.0pt; mso-border-right-themecolor: text1; mso-border-top-alt: .5pt; mso-border-top-themecolor: text1; mso-border-left-alt: .5pt; mso-border-left-themecolor: text1; mso-border-alt: .5pt; mso-border-themecolor: text1; padding: 0in 5.4pt 0in 5.4pt;" valign="top" width="111">
<p class="MsoNormal">3 mls</p>
</td>
</tr>
<tr style="mso-yfti-irow: 2;">
<td style="width: 110.7pt; border: 1.0pt; mso-border-themecolor: text1; border-top: none; mso-border-top-alt: .5pt; mso-border-top-themecolor: text1; mso-border-alt: .5pt; padding: 0in 5.4pt 0in 5.4pt;" valign="top" width="111">
<p class="MsoNormal">NaCl</p>
</td>
<td style="width: 110.7pt; border-top: none; border-left: none; border-bottom: 1.0pt; mso-border-bottom-themecolor: text1; border-right: 1.0pt; mso-border-right-themecolor: text1; mso-border-top-alt: .5pt; mso-border-top-themecolor: text1; mso-border-left-alt: .5pt; mso-border-left-themecolor: text1; mso-border-alt: .5pt; mso-border-themecolor: text1; padding: 0in 5.4pt 0in 5.4pt;" valign="top" width="111">
<p class="MsoNormal">5M</p>
</td>
<td style="width: 110.7pt; border-top: none; border-left: none; border-bottom: 1.0pt; mso-border-bottom-themecolor: text1; border-right: 1.0pt; mso-border-right-themecolor: text1; mso-border-top-alt: .5pt; mso-border-top-themecolor: text1; mso-border-left-alt: .5pt; mso-border-left-themecolor: text1; mso-border-alt: .5pt; mso-border-themecolor: text1; padding: 0in 5.4pt 0in 5.4pt;" valign="top" width="111">
<p class="MsoNormal">100mM (10mM)</p>
</td>
<td style="width: 110.7pt; border-top: none; border-left: none; border-bottom: 1.0pt; mso-border-bottom-themecolor: text1; border-right: 1.0pt; mso-border-right-themecolor: text1; mso-border-top-alt: .5pt; mso-border-top-themecolor: text1; mso-border-left-alt: .5pt; mso-border-left-themecolor: text1; mso-border-alt: .5pt; mso-border-themecolor: text1; padding: 0in 5.4pt 0in 5.4pt;" valign="top" width="111">
<p class="MsoNormal">200ul</p>
</td>
</tr>
<tr style="mso-yfti-irow: 3;">
<td style="width: 110.7pt; border: 1.0pt; mso-border-themecolor: text1; border-top: none; mso-border-top-alt: .5pt; mso-border-top-themecolor: text1; mso-border-alt: .5pt; padding: 0in 5.4pt 0in 5.4pt;" valign="top" width="111">
<p class="MsoNormal">EDTA</p>
</td>
<td style="width: 110.7pt; border-top: none; border-left: none; border-bottom: 1.0pt; mso-border-bottom-themecolor: text1; border-right: 1.0pt; mso-border-right-themecolor: text1; mso-border-top-alt: .5pt; mso-border-top-themecolor: text1; mso-border-left-alt: .5pt; mso-border-left-themecolor: text1; mso-border-alt: .5pt; mso-border-themecolor: text1; padding: 0in 5.4pt 0in 5.4pt;" valign="top" width="111">
<p class="MsoNormal">0.5M (pH-8.0)</p>
</td>
<td style="width: 110.7pt; border-top: none; border-left: none; border-bottom: 1.0pt; mso-border-bottom-themecolor: text1; border-right: 1.0pt; mso-border-right-themecolor: text1; mso-border-top-alt: .5pt; mso-border-top-themecolor: text1; mso-border-left-alt: .5pt; mso-border-left-themecolor: text1; mso-border-alt: .5pt; mso-border-themecolor: text1; padding: 0in 5.4pt 0in 5.4pt;" valign="top" width="111">
<p class="MsoNormal">1mM (0.1mM)</p>
</td>
<td style="width: 110.7pt; border-top: none; border-left: none; border-bottom: 1.0pt; mso-border-bottom-themecolor: text1; border-right: 1.0pt; mso-border-right-themecolor: text1; mso-border-top-alt: .5pt; mso-border-top-themecolor: text1; mso-border-left-alt: .5pt; mso-border-left-themecolor: text1; mso-border-alt: .5pt; mso-border-themecolor: text1; padding: 0in 5.4pt 0in 5.4pt;" valign="top" width="111">
<p class="MsoNormal">10ul</p>
</td>
</tr>
<tr style="mso-yfti-irow: 4;">
<td style="width: 110.7pt; border: 1.0pt; mso-border-themecolor: text1; border-top: none; mso-border-top-alt: .5pt; mso-border-top-themecolor: text1; mso-border-alt: .5pt; padding: 0in 5.4pt 0in 5.4pt;" valign="top" width="111">
<p class="MsoNormal">EGTA</p>
</td>
<td style="width: 110.7pt; border-top: none; border-left: none; border-bottom: 1.0pt; mso-border-bottom-themecolor: text1; border-right: 1.0pt; mso-border-right-themecolor: text1; mso-border-top-alt: .5pt; mso-border-top-themecolor: text1; mso-border-left-alt: .5pt; mso-border-left-themecolor: text1; mso-border-alt: .5pt; mso-border-themecolor: text1; padding: 0in 5.4pt 0in 5.4pt;" valign="top" width="111">
<p class="MsoNormal">0.1M (pH-8.0)</p>
</td>
<td style="width: 110.7pt; border-top: none; border-left: none; border-bottom: 1.0pt; mso-border-bottom-themecolor: text1; border-right: 1.0pt; mso-border-right-themecolor: text1; mso-border-top-alt: .5pt; mso-border-top-themecolor: text1; mso-border-left-alt: .5pt; mso-border-left-themecolor: text1; mso-border-alt: .5pt; mso-border-themecolor: text1; padding: 0in 5.4pt 0in 5.4pt;" valign="top" width="111">
<p class="MsoNormal">500uM (50uM)</p>
</td>
<td style="width: 110.7pt; border-top: none; border-left: none; border-bottom: 1.0pt; mso-border-bottom-themecolor: text1; border-right: 1.0pt; mso-border-right-themecolor: text1; mso-border-top-alt: .5pt; mso-border-top-themecolor: text1; mso-border-left-alt: .5pt; mso-border-left-themecolor: text1; mso-border-alt: .5pt; mso-border-themecolor: text1; padding: 0in 5.4pt 0in 5.4pt;" valign="top" width="111">
<p class="MsoNormal">50ul</p>
</td>
</tr>
<tr style="mso-yfti-irow: 5;">
<td style="width: 110.7pt; border: 1.0pt; mso-border-themecolor: text1; border-top: none; mso-border-top-alt: .5pt; mso-border-top-themecolor: text1; mso-border-alt: .5pt; padding: 0in 5.4pt 0in 5.4pt;" valign="top" width="111">
<p class="MsoNormal">HEPES</p>
</td>
<td style="width: 110.7pt; border-top: none; border-left: none; border-bottom: 1.0pt; mso-border-bottom-themecolor: text1; border-right: 1.0pt; mso-border-right-themecolor: text1; mso-border-top-alt: .5pt; mso-border-top-themecolor: text1; mso-border-left-alt: .5pt; mso-border-left-themecolor: text1; mso-border-alt: .5pt; mso-border-themecolor: text1; padding: 0in 5.4pt 0in 5.4pt;" valign="top" width="111">
<p class="MsoNormal">1M (pH-7.5)</p>
</td>
<td style="width: 110.7pt; border-top: none; border-left: none; border-bottom: 1.0pt; mso-border-bottom-themecolor: text1; border-right: 1.0pt; mso-border-right-themecolor: text1; mso-border-top-alt: .5pt; mso-border-top-themecolor: text1; mso-border-left-alt: .5pt; mso-border-left-themecolor: text1; mso-border-alt: .5pt; mso-border-themecolor: text1; padding: 0in 5.4pt 0in 5.4pt;" valign="top" width="111">
<p class="MsoNormal">50mM (5mM)</p>
</td>
<td style="width: 110.7pt; border-top: none; border-left: none; border-bottom: 1.0pt; mso-border-bottom-themecolor: text1; border-right: 1.0pt; mso-border-right-themecolor: text1; mso-border-top-alt: .5pt; mso-border-top-themecolor: text1; mso-border-left-alt: .5pt; mso-border-left-themecolor: text1; mso-border-alt: .5pt; mso-border-themecolor: text1; padding: 0in 5.4pt 0in 5.4pt;" valign="top" width="111">
<p class="MsoNormal">500ul</p>
</td>
</tr>
<tr style="mso-yfti-irow: 6; mso-yfti-lastrow: yes;">
<td style="width: 110.7pt; border: 1.0pt; mso-border-themecolor: text1; border-top: none; mso-border-top-alt: .5pt; mso-border-top-themecolor: text1; mso-border-alt: .5pt; padding: 0in 5.4pt 0in 5.4pt;" valign="top" width="111">
<p class="MsoNormal">Water</p>
</td>
<td style="width: 110.7pt; border-top: none; border-left: none; border-bottom: 1.0pt; mso-border-bottom-themecolor: text1; border-right: 1.0pt; mso-border-right-themecolor: text1; mso-border-top-alt: .5pt; mso-border-top-themecolor: text1; mso-border-left-alt: .5pt; mso-border-left-themecolor: text1; mso-border-alt: .5pt; mso-border-themecolor: text1; padding: 0in 5.4pt 0in 5.4pt;" valign="top" width="111">
<p class="MsoNormal"> </p>
</td>
<td style="width: 110.7pt; border-top: none; border-left: none; border-bottom: 1.0pt; mso-border-bottom-themecolor: text1; border-right: 1.0pt; mso-border-right-themecolor: text1; mso-border-top-alt: .5pt; mso-border-top-themecolor: text1; mso-border-left-alt: .5pt; mso-border-left-themecolor: text1; mso-border-alt: .5pt; mso-border-themecolor: text1; padding: 0in 5.4pt 0in 5.4pt;" valign="top" width="111">
<p class="MsoNormal"> </p>
</td>
<td style="width: 110.7pt; border-top: none; border-left: none; border-bottom: 1.0pt; mso-border-bottom-themecolor: text1; border-right: 1.0pt; mso-border-right-themecolor: text1; mso-border-top-alt: .5pt; mso-border-top-themecolor: text1; mso-border-left-alt: .5pt; mso-border-left-themecolor: text1; mso-border-alt: .5pt; mso-border-themecolor: text1; padding: 0in 5.4pt 0in 5.4pt;" valign="top" width="111">
<p class="MsoNormal">6.2ml</p>
</td>
</tr>
</tbody>
</table>