|
|
| Line 103: |
Line 103: |
| * Add 100 μL streptavidin conjugated to HRP | | * Add 100 μL streptavidin conjugated to HRP |
| * Incubate 25 min. @ RT | | * Incubate 25 min. @ RT |
| | * Remove liquid |
| | * Wash 5x 200 μL Washing buffer |
| | ** {{todo| prepare TBM substrate}} |
| | * Add 100 μL substrate |
| | * Incubate 30 min. @ RT |
| | * {{todo|}}Stop reaction, add 200 μL... |
| <!--{| border="1" | | <!--{| border="1" |
| |+'''96-wells plate''' | | |+'''96-wells plate''' |
Revision as of 07:48, 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
<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>
|
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
- Streptavidin conjugated to HRP
- 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
- 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
- Add 100 μL substrate
- Incubate 30 min. @ RT
- Stop reaction, add 200 μL...
Notes
- Of the 96-wells plate well F1 contain a Polystyrene fragment
Results
Conclusion
Related
|
ELISA
