User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/02/18: Difference between revisions

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* Washing buffer
* Washing buffer
* IL-8 antibody
* IL-8 antibody
 
* Substrate buffer
** 1.5 sodiumacetate, resuspend in 80 mL ultrapure water
** pH 5.5
** up to 100 mL with ultrapure water
* TMB stock solution (light sensitive)
** 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]


===Method===
===Method===

Revision as of 06:45, 18 February 2010

<html><style type="text/css"> .todo { color: red } .done { color: green} </style></html>

ELISA <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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Summary

  • Short summary

Materials & Methods

Materials

  • Coating buffer
  • IL-8 antibody
  • 96-wells plate (MaxiSorb)
  • PBS
  • BSA
  • Pelikine Compact™ Human IL-8 ELISA kit
  • Washing buffer
  • IL-8 antibody
  • Substrate buffer
    • 1.5 sodiumacetate, resuspend in 80 mL ultrapure water
    • pH 5.5
    • up to 100 mL with ultrapure water
  • TMB stock solution (light sensitive)

Method

Coating of plate

  • Mix
    • 24 mL coating buffer
    • 80 μL primary antibody
  • Add 100 μL per well (96-wells plate)
  • Incubate ON

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
  • Was 5x 200 μL Washing buffer
  • Remove buffer
    • prepare biotinylated antibody
  • Add 100 μL Biotinylated antibody
  • Incubate 1h @ RT

Notes

  • Of the 96-wells plate well F1 contain a Polystyrene fragment

Results

  • WHAT?

Conclusion

  • DONE Ow..

Related



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