User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/02/24: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Co- | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Co-immunoprecipitation part II, (Stimulation hTERT cells v2)</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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** 1.06 mg β-glycerolphosphate | ** 1.06 mg β-glycerolphosphate | ||
** 1 mL [http://wikilaboratory.com/index.php?title=RIPA_Buffer RIPA buffer] | ** 1 mL [http://wikilaboratory.com/index.php?title=RIPA_Buffer RIPA buffer] | ||
** 1 μL Apoprotein | ** 1 μL Apoprotein (1 mg/mL) | ||
** 1 μL Leupeptin | ** 1 μL Leupeptin (1 mg/mL) | ||
** 1 μL Pepstatin (A) | ** 1 μL Pepstatin (A) (1 mg/mL) | ||
** 5 μL Na<sub>3</sub>VO<sub>4</sub> | ** 5 μL Na<sub>3</sub>VO<sub>4</sub> | ||
** 5 μL NaF | ** 5 μL NaF (200 mM) | ||
* [[PBS]] | * [[PBS]] | ||
*[http://www.piercenet.com/Products/Browse.cfm?fldID=02020105 Coomassie (Bradford) Protein Assay Kit] | *[http://www.piercenet.com/Products/Browse.cfm?fldID=02020105 Coomassie (Bradford) Protein Assay Kit] | ||
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===Method=== | ===Method=== | ||
====Determination of protein concentration==== | ====[http://genome.med.yale.edu/images/a/aa/BCA.pdf Determination of protein concentration]==== | ||
* Use cells put on S<sup>0</sup> [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/23| 23Feb2010]] | * Use cells put on S<sup>0</sup> [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/23| 23Feb2010]] | ||
* Put cells on ice | * Put cells on ice | ||
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# Beads w. antibody (30 μL) & sample (200 μL) | # Beads w. antibody (30 μL) & sample (200 μL) | ||
(possible also beads (30 μL) with only sample (200 μL)) | (possible also beads (30 μL) with only sample (200 μL)) | ||
* Incubate ON @ °C | * Incubate ON @ 4 °C | ||
===Stimulation hTERT (BSc students=== | |||
* HT31P dose response curve (0 - 50 μM) | |||
* HT31 (200 μM) /S<sup>0</sup> Basal | |||
==Results== | ==Results== | ||
{| border="1" | {| border="1" | ||
|+'''Standard curve | |+'''Standard curve Pierce Assay''' | ||
| | | | ||
!Prot. Conc. (μg/mL) | !Prot. Conc. (μg/mL) | ||
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|} | |} | ||
==Related== | ===Related entries=== | ||
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/19| | ====Run 2: Ht31 Dose/Effect Curve & S<sup>0</sup> D9/D12==== | ||
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/23| Putting | *[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/19|Counting cells 19Feb2010]] | ||
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/23|Putting to S<sup>0</sup> 23Feb2010]] | |||
===Same actions=== | ===Same actions=== | ||
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/16| Stimulation hTERT cells]] | *[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/16| Stimulation hTERT cells]] | ||
==Related topics== | ==Related topics== | ||
* [[Cell_and_tissue_lysis_hub]] | * [[Cell_and_tissue_lysis_hub]] | ||
Revision as of 08:02, 23 March 2010
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 Co-immunoprecipitation part II, (Stimulation hTERT cells v2)
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SummaryConditions
With a dose response range of 0 - 50 μg/mLug Ht31, instead of HT31P S0 was used. Materials & MethodsMaterials
MethodDetermination of protein concentration
Co-immunoprecipitation
(possible also beads (30 μL) with only sample (200 μL))
Stimulation hTERT (BSc students
Results
Related entriesRun 2: Ht31 Dose/Effect Curve & S0 D9/D12Same actionsRelated topics | ||||||||||||||||||||||||||||||||||||||||||||||||||||||
