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ELISA
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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))
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1
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2
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3
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4
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5
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6
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A
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100/100
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100/100
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100/100
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100/100
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100/100
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100/100
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B
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150/50
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150/50
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150/50
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175/25
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175/25
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175/25
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C
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150/50
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150/50
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150/50
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175/25
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175/25
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175/25
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D
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150/50
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150/50
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150/50
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175/25
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175/25
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175/25
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- 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
Conclusion
Related
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