User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/02/18: Difference between revisions
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===Materials=== | ===Materials=== | ||
* Coating buffer | * Coating buffer | ||
* IL-8 antibody | * IL-8 antibody Coating antibody | ||
* 96-wells plate (MaxiSorb) | * 96-wells plate (MaxiSorb) | ||
* [[PBS]] | * [[PBS]] 5% | ||
* [http://en.wikipedia.org/wiki/Bovine_serum_albumin BSA] | * [http://en.wikipedia.org/wiki/Bovine_serum_albumin BSA] 5% (in PBS) | ||
* [http://www.sanquin.pl/datasheet/m1918.pdf Pelikine Compact™ Human IL-8 ELISA kit] | * [http://www.sanquin.pl/datasheet/m1918.pdf Pelikine Compact™ Human IL-8 ELISA kit] | ||
* Washing buffer | * Washing buffer (PBS + 0.005% TWEEN 20) | ||
* IL-8 antibody | * IL-8 antibody | ||
* Streptavidin conjugated to HRP | * Streptavidin conjugated to HRP | ||
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** 30 mg [http://en.wikipedia.org/wiki/3,3%E2%80%99,5,5%E2%80%99-Tetramethylbenzidine TMB] | ** 30 mg [http://en.wikipedia.org/wiki/3,3%E2%80%99,5,5%E2%80%99-Tetramethylbenzidine TMB] | ||
** 5 mL [http://en.wikipedia.org/wiki/Dimethyl_sulfoxide DMSO] | ** 5 mL [http://en.wikipedia.org/wiki/Dimethyl_sulfoxide DMSO] | ||
===Method=== | ===Method=== | ||
<b><u>Coating of plate</u></b> | <b><u>Coating of plate</u></b> | ||
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* Wash 5x 200 μL Washing buffer | * Wash 5x 200 μL Washing buffer | ||
** {{todo| prepare TBM substrate}} | ** {{todo| prepare TBM substrate}} | ||
** 1.2 μL H<sub>2</sub>O | ** 1.2 μL H<sub>2</sub>O<sub>2</sub> (30%) | ||
** 400 μL TBM stock solution | ** 400 μL TBM stock solution | ||
** 24 mL substrate buffer | ** 24 mL substrate buffer | ||
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* Stop reaction, add 100 μL stopping solution | * Stop reaction, add 100 μL stopping solution | ||
** It will turn yellow | ** It will turn yellow | ||
* Measure absorbance @ | * Measure absorbance @ 450 nm | ||
** Check duplo standard curve | ** Check duplo standard curve | ||
** Check if values fall within standard curve (preferred) | ** Check if values fall within standard curve (preferred) | ||
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*When comparing the effect of CSE without (HT31P) and with (HT31) PKA/AKAP interaction inhibition, it seems that when AKAP/PKA association is inhibited it decreases CSE induced IL-8 release. | *When comparing the effect of CSE without (HT31P) and with (HT31) PKA/AKAP interaction inhibition, it seems that when AKAP/PKA association is inhibited it decreases CSE induced IL-8 release. | ||
** PKA/AKAP interaction is required for CSE induced IL-8 secretion. | ** PKA/AKAP interaction is required for CSE induced IL-8 secretion. | ||
* When inducing Epac no effect is seen when comparing the | * When inducing Epac no effect is seen when comparing the HT31 situation, however slight decrease of IL-8 production is seen when HT31P control situation receives Epac stimulation. | ||
** When Epac can compete with PKA for the present cAMP it inhibits the increased IL-8 secretion. | ** When Epac can compete with PKA for the present cAMP it inhibits the increased IL-8 secretion. | ||
** Epac does not have an influence when PKA can not have its effect | ** Epac does not have an influence when PKA can not have its effect | ||
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*<b> PKA is the more important effector of cAMP compared to Epac</b> | *<b> PKA is the more important effector of cAMP compared to Epac</b> | ||
==Related== | ==Related entries== | ||
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/10| Splitting cells]] | ===Run 1: Ht31/Ht31P D9/D12=== | ||
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/12| Counting cells]] | *[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/10|Splitting cells 10Feb2010]] | ||
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/15| Putting | *[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/12|Counting cells 12Feb2010]] | ||
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/16| | *[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/15|Putting to S<sup>0</sup> 15Feb2010]] | ||
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/17| Viability | *[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/16|Stimulating cells 16Feb2010]] | ||
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/17|Ending stimulation & Viability 17Feb2010]] | |||
==Attachment== | |||
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</html> [[Media:HTERT_Donor_9_ht31_180210.xls| Excel hTERT D9]] | |||
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<a href="http://openwetware.org/wiki/Image:HTERT_Donor_12_ht31_160310.xls"> | |||
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</html> [[Media:HTERT_Donor_12_ht31_180210.xls| Excel hTERT D12]] | |||
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Revision as of 01:19, 26 March 2010
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ELISA | <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> | |||||||||||||||||||||||||||||||||||
SummaryTo determine the amount of interleukin 8 (IL-8) produced by the airway smooth muscle cells (ASMC's) ELISA is performed using the PeliKine Compact™ human IL-8 ELISA kit. In this way the effect of PKA, EPAC and CSE on the inflammatory cytokine can be determined. Materials & MethodsMaterials
MethodCoating of plate
Preparing Sandwich
Notes
Results
Conclusion
Related entriesRun 1: Ht31/Ht31P D9/D12
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</html> Excel hTERT D9
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