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

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Summary

  • ELISA
    • Samples 17-3-10
    • Samples 24-3-10

Materials & Methods

Materials

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))


24-wells (dilution buffer (μL)/sample (μL))
1 2 3 4 5 6
A 100/100 100/100 100/100 100/100 100/100 100/100
B 150/50 150/50 150/50 175/25 175/25 175/25
C 150/50 150/50 150/50 175/25 175/25 175/25
D 150/50 150/50 150/50 175/25 175/25 175/25


  • 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
    • prepare biotinylated antibody
  • 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
    • prepare TBM substrate
    • 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)


ELISA Plate #1 D9
01 02 03 04 05 06 07 08 09 10 11 12
A 1A1 1A2 1A3 1A4 1A5 1A6 2A1 2A2 2A3 2A4 2A5 2A6
B 1B1 1B2 1B3 1B4 1B5 1B6 2B1 2B2 2B3 2B4 2B5 2B6
C 1C1 1C2 1C3 1C4 1C5 1C6 2C1 2C2 2C3 2C4 2C5 2C6
D 1D1 1D2 1D3 1D4 1D5 1D6 2D1 2D2 2D3 2D4 2D5 2D6
E Tonio Tonio Tonio Tonio Tonio Tonio Tonio Tonio 240 96.0 38.4 15.4
F Tonio Tonio Tonio Tonio Tonio Tonio Tonio Tonio 240 96.0 38.4 15.4
G Tonio Tonio Tonio Tonio Tonio Tonio Tonio Tonio 6.1 2.5 1.0 0.0
H Tonio Tonio Tonio Tonio Tonio Tonio Tonio Tonio 6.1 2.5 1.0 0.0


ELISA Plate #2 D12
01 02 03 04 05 06 07 08 09 10 11 12
A 1A1 1A2 1A3 1A4 1A5 1A6 2A1 2A2 2A3 2A4 2A5 2A6
B 1B1 1B2 1B3 1B4 1B5 1B6 2B1 2B2 2B3 2B4 2B5 2B6
C 1C1 1C2 1C3 1C4 1C5 1C6 2C1 2C2 2C3 2C4 2C5 2C6
D 1D1 1D2 1D3 1D4 1D5 1D6 2D1 2D2 2D3 2D4 2D5 2D6
E Tonio Tonio Tonio Tonio Tonio Tonio Tonio Tonio 240 96.0 38.4 15.4
F Tonio Tonio Tonio Tonio Tonio Tonio Tonio Tonio 240 96.0 38.4 15.4
G Tonio Tonio Tonio Tonio Tonio Tonio Tonio Tonio 6.1 2.5 1.0 0.0
H Tonio Tonio Tonio Tonio Tonio Tonio Tonio Tonio 6.1 2.5 1.0 0.0

Results

Ht31P toxicity

Ht31PtoxicityIL8secretionD9P23250310.JPG Ht31PtoxicityIL8secretionD12P27250310.JPG

Effect of PKA/Epac stimulation

IL8secretionD9P25250310.JPG IL8secretionD12P29250310.JPG

Compilation

IL8secretionD9&D12250310.jpg

T.TOETS S0 & Ht31 ' ' ' ' ' ' '
S0 p = CSE (S0) p = CSE (Ht31) p =
CTR vs CSE 0,046 <0.05 vs CTR (Ht31) 0,052 - vs CTR (S0) 0,020 <0.05
CTR vs CSE+8p 0,027 <0.05 vs CSE+8P (Ht31) 0,271 - vs CSE+8P (S0) 0,146 -
CTR vs CSE+6Bnz 0,053 - vs CSE+6Bnz (Ht31 0,231 - vs CSE+6Bnz (S0) 0,029 <0.05
CSE vs CSE+8p 0,633 -
CSE vs CSE+6Bnz 0,080 -
CSE+8p vs CSE+6Bnz 0,062 - CSE+8p (S0) CSE+8p (Ht31)
Ht31 vs CTR (Ht31) 0,032 <0.05 vs CTR (S0) 0,027 <0.05
CTR vs CSE 0,022 <0.05 vs CSE+6Bnz (Ht31 0,324 - vs CSE+6Bnz (S0) 0,038 <0.05
CTR vs CSE+8p 0,029 <0.05
CTR vs CSE+6Bnz 0,079 -
CSE vs CSE+8p 0,868 - CSE+6Bnz (S0) CSE+6Bnz (Ht31)
CSE vs CSE+6Bnz 0,058 - vs CTR (Ht31) 0,115 - vs CTR (S0) 0,060 -
CSE+8p vs CSE+6Bnz 0,068 -
S0 vs Ht31
CTR 0,242 -
CSE 0,298 -
CSE+8p 0,148 -
CSE+6Bnz 0,206 -

Notes

  • CSE + Ht31P (0 μM) of D9 (17March2010) got pipetted into the ELISA well instead of the intended CSE+8pcpt & S0 of D9 (24March2010)
    • Sample was removed and replaced by the intended sample
    • Dilution of sample was deemed enough to not expect large contamination

Conclusion

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

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