User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/03/15: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Co-IP & ELISA prep</span> | ||
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html> </html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}} | |style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html> </html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}} | ||
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**Take 70 mL of Buffer A | **Take 70 mL of Buffer A | ||
**Add Buffer B until pH of 9.6 has reached (150 - 200 mL) | **Add Buffer B until pH of 9.6 has reached (150 - 200 mL) | ||
* IL-8 coating antibody | |||
* 96-wells plate (MaxiSorb) | |||
===Method=== | ===Method=== | ||
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*** 80 μL for Only Beads (D9/D12) | *** 80 μL for Only Beads (D9/D12) | ||
*** (30 μL for each condition) | *** (30 μL for each condition) | ||
** Add 1:100 antibody (or not for control), 2 μL | ** Add 1:100 (PKA RIIβ) antibody (or not for control), 2 μL | ||
** Incubate ON @ 4 °C | ** Incubate ON @ 4 °C | ||
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* Add 100 μL per well (96-wells plate) | * Add 100 μL per well (96-wells plate) | ||
* Incubate ON | * Incubate ON | ||
==Notes== | ==Notes== | ||
* 6-wells plates which had their medium refreshed [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/12| 12March2010]] were thrown out | |||
* In putting cells to S<sup>0</sup> D12 well A2 got 1 mL instead of 0.5 mL DMEM S<sup>0</sup>, no large complications are to be expected. | |||
* Cells used for Co-IP and those put to S<sup>0</sup> were | |||
** D9 P23, plated out [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/12|12March2010]] | |||
** D12 P27, plated out [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/12|12March2010]] | |||
* For the Pierce determination new vials B and C of the standard curve were prepared. | |||
==Results== | ==Results== | ||
* | *Pierce determination Co-IP | ||
** D9: 2.43 mg/mL | |||
** D12: 2.10 mg/mL | |||
==Conclusion== | ==Conclusion== | ||
* | *There is enough protein present for Co-IP (>1mg/mL) | ||
Revision as of 12:02, 15 March 2010
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Co-IP & ELISA prep | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> |
Summary
Materials & MethodsMaterials
MethodPutting cells to S0
Determination of protein concentration
Co-immunoprecipitation part I
Coating of ELISA plate
Notes
Results
Conclusion
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