User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/03/25

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] ELISA
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


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Summary

 * ELISA
 * Samples 17-3-10
 * Samples 24-3-10

Materials

 * See 18Feb2010

Method
 Preparing Sandwich   
 * Wash coated plate 5x 200 μL PBS
 * Remove PBS
 * Add 200 μL BSA
 * Incubate 1h @ RT and remove BSA
 * Dilute samples as shown below for each 24-wells (dilution buffer (μL)/sample (μL))
 * Wash of BSA with 200 μL Washing buffer 5x
 * Add 100 μL of diluted sample
 * Incubate ≥1h @ RT
 * Wash 5x 200 μL Washing buffer
 * Remove buffer
 * Add 100 μL Biotinylated antibody
 * Incubate 1h @ RT
 * Remove antibody
 * Wash 5x 200 μL Washing buffer
 * Add 100 μL streptavidin conjugated to HRP
 * Incubate 25 min. @ RT
 * Remove liquid
 * Wash 5x 200 μL Washing buffer
 * 1.2 μL H2O
 * 400 μL TBM stock solution
 * 24 mL substrate buffer
 * Add 100 μL substrate
 * Incubate 30 min. @ RT
 * Stop reaction, add 100 μL stopping solution
 * It will turn yellow
 * Measure absorbance @ 450 nm
 * Check duplo standard curve
 * Check if values fall within standard curve (preferred)
 * Check duplo standard curve
 * Check if values fall within standard curve (preferred)

Compilation

 * Combined with statistics 18March2010

Ht31P toxicity

 * Ht31P is not usable in hTERT cells due to the too low tolerance, leading to high cell mortality and a great effect on the IL-8 production

Epac/PKA stimulation

 * Significance decreased due to the addition of the new values, instead of the expected increase however the figures should be looked at more closely
 * SEM is more accurate to specify deviation between samples, compared to StDev for population research
 * D12 shows much larger IL-8 secretion compared to D9
 * However the same trend is seen between different treatments
 * Disturbing PKA/AKAP interactions increases the IL-8 secretion
 * Stimulation of Epac does not seem to influence the IL-8 secretion
 * Stimulation of PKA seems to counteract the negative influence of the PKA/AKAP inhibition, and decreases IL-8 secretion

Related entries

 * Ht31P toxicity ELISA Samples
 * Stimulation Ht31P 16March2010
 * Viability staining 24March2010
 * Stimulation Ht31/S0
 * Statistical Analysis 18March2010

Same actions

 * ELISA 18Feb2010
 * ELISA 26Feb2010
 * ELISA 16March2010
 * Statistical Analysis 18March2010

Attachment
   [[Media:HTERT_Donor_9_P23_%26_P25_240310.xls|Excel hTERT D9 P23 & P25]]    [[Media:HTERT_Donor_12_P27_%26_P29_240310.xls|Excel hTERT D12 P27 & P29]]


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