Team:Warsaw/Calendar-Main/8 September 2008

From 2008.igem.org

(Difference between revisions)
 
(11 intermediate revisions not shown)
Line 4: Line 4:
<html>
<html>
 +
<h3>Our webpage has a completely new layout</h3>
 +
<h4>Emilia</h4>
-
<h3>Purification of proteins: A-alpha, Z-alpha and Z-omega</h3><h4>Piotr</h4>
+
<h3>Purification of proteins: A-alpha</h3><h4>Piotr</h4>
-
<p>Electrophoresis in polyacrylamide gel (12 %) of purified Z-omega and Z-alpha.</p>
+
<p>Electrophoresis in polyacrylamide gel (12 %) of sonicate containing A-alpha (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/8_September_2008#fig1">Fig. 1.</a>).</p>
 +
<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/4/49/September_8_th.jpg" width=150 /></a><var><b>Fig. 1.</b> Sonicate containing A-alpha protein fusion<br>
 +
The arrow shows place of our overexpressed protein:<br>
 +
1. Marker<br>
 +
2. sonicate A-alpha with induction<br></var>
-
<h3>Purification of A-alpha with His-tag.</h3>
+
 
 +
 
 +
 
 +
<h3></h3>
<h4>Michał L.</h4>
<h4>Michał L.</h4>
<ol>
<ol>
-
<li>200 ml of overnight E. coli culture was spun down 6000 RPM, 10 min at 4&deg;C</li>
+
 
-
<li>The pelet was resuspended in 20 ml of sonication buffer A (50 mM phosphate buffer pH 7.2, 100 mM NaCl, 10 mM imidazole) </li>
+
<li>Supernatant containing A-alpha was mixed with NiNta bead in proportion 10:1 and placed on rotating platform for 30 min at 4&deg;C</li>
-
<li>The mixture was sonicated in 20 pulses: 90 W pulse duration - 20 s, pause 40 s. </li>
+
<li>Bead was spun down at 6000 RPM, 5 min at 4&deg;C</li>
-
<li>The post-sonication debris were spun down 13200 RPM, 20 min at 4&deg;C</li>
+
<li>Supernatant was discarded and equal amount of Elution buffer 1 (Sonication buffer A suplemented with 50 mM imidazole) was placed on top of the bead</li>
-
<li>Clear supernatant was mixed with NiNta bead in proportion 10:1 and placed on rotating platform for 30 min at 4&deg;C</li>
+
<li>Bead was spun down at 6000 RPM, 5 min at 4&deg;C</li>
-
<li>The bead was spun down 6000 RPM, 5 min at 4&deg;C</li>
+
<li>Supernatant was collected for further analysis and equal amount of Elution buffer 2 (Sonication buffer A supplemented with 100 mM imidazole) was placed on top of the bead</li>
-
<li>The supernatant was discarded and equal amount of Elution buffer 1 (Sonication buffer A suplemented with 50 mM imidazole) was placed on top of the bead</li>
+
<li>The above two steps were repeated for elution buffers 3 and 4 (150 mM and 200 mM imidazole)</li>
-
<li>The bead was spun down 6000 RPM, 5 min at 4&deg;C</li>
+
<li>All fractions obtained in this procedure were resolved on 8% SDS-PAGE gel. It turned out that optimum imidazole concentration for elution of His-tagged A protein is 150 mM</li>
-
<li>The supernatant was collected for further analysis and equal amount of Elution buffer 2 (Sonication buffer A suplemented with 100 mM imidazole) was placed on top of the bead</li>
+
-
<li>Above two steps were repeated for elution buffers 3 and 4 (150mM and 200 mM imidazole)</li>
+
-
<li>All fractions obtained in this procedure were resolved on 8% SDS-PAGE gel. It turned out that optimum imidazole concentration for elution of HisTagged A protein is 150 mM</li>
+
<ol>
<ol>
 +
</html>
</html>
<!-- do not remove this! -->
<!-- do not remove this! -->
{{WarNotebookEnd}}
{{WarNotebookEnd}}

Latest revision as of 19:38, 26 October 2008

Gallery Bricks Notebook Team Project Home


Previous day
return to main notebook page
Previous entry
Interactive list of topics
next notebook entry

 


Our webpage has a completely new layout

Emilia

Purification of proteins: A-alpha

Piotr

Electrophoresis in polyacrylamide gel (12 %) of sonicate containing A-alpha (Fig. 1.).

Fig. 1. Sonicate containing A-alpha protein fusion
The arrow shows place of our overexpressed protein:
1. Marker
2. sonicate A-alpha with induction

Michał L.

  1. Supernatant containing A-alpha was mixed with NiNta bead in proportion 10:1 and placed on rotating platform for 30 min at 4°C
  2. Bead was spun down at 6000 RPM, 5 min at 4°C
  3. Supernatant was discarded and equal amount of Elution buffer 1 (Sonication buffer A suplemented with 50 mM imidazole) was placed on top of the bead
  4. Bead was spun down at 6000 RPM, 5 min at 4°C
  5. Supernatant was collected for further analysis and equal amount of Elution buffer 2 (Sonication buffer A supplemented with 100 mM imidazole) was placed on top of the bead
  6. The above two steps were repeated for elution buffers 3 and 4 (150 mM and 200 mM imidazole)
  7. All fractions obtained in this procedure were resolved on 8% SDS-PAGE gel. It turned out that optimum imidazole concentration for elution of His-tagged A protein is 150 mM

Retrieved from "http://2008.igem.org/Team:Warsaw/Calendar-Main/8_September_2008"