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 ELISA
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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))
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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
- 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
- Measure absorbance @ 450 nm
- Check duplo standard curve
- Check if values fall within standard curve (preferred)
ELISA Plate #1 D9
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01
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02
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03
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04
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05
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06
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07
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08
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09
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10
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11
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12
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| A
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1A1
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1A2
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1A3
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1A4
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1A5
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1A6
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2A1
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2A2
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2A3
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2A4
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2A5
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2A6
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| B
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1B1
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1B2
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1B3
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1B4
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1B5
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1B6
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2B1
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2B2
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2B3
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2B4
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2B5
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2B6
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| C
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1C1
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1C2
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1C3
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1C4
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1C5
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1C6
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2C1
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2C2
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2C3
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2C4
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2C5
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2C6
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| D
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1D1
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1D2
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1D3
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1D4
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1D5
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1D6
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2D1
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2D2
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2D3
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2D4
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2D5
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2D6
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| E
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Tonio
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Tonio
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Tonio
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Tonio
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Tonio
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Tonio
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Tonio
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Tonio
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240
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96.0
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38.4
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15.4
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| F
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Tonio
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Tonio
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Tonio
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Tonio
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Tonio
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Tonio
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Tonio
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Tonio
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240
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96.0
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38.4
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15.4
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| G
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Tonio
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Tonio
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Tonio
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Tonio
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Tonio
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Tonio
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Tonio
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Tonio
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6.1
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2.5
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1.0
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0.0
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| H
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Tonio
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Tonio
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Tonio
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Tonio
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Tonio
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Tonio
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Tonio
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Tonio
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6.1
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2.5
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1.0
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0.0
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ELISA Plate #2 D12
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01
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02
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03
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04
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05
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06
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07
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08
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09
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10
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11
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12
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| A
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1A1
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1A2
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1A3
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1A4
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1A5
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1A6
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2A1
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2A2
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2A3
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2A4
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2A5
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2A6
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| B
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1B1
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1B2
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1B3
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1B4
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1B5
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1B6
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2B1
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2B2
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2B3
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2B4
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2B5
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2B6
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| C
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1C1
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1C2
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1C3
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1C4
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1C5
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1C6
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2C1
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2C2
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2C3
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2C4
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2C5
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2C6
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| D
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1D1
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1D2
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1D3
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1D4
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1D5
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1D6
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2D1
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2D2
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2D3
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2D4
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2D5
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2D6
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| E
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Tonio
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Tonio
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Tonio
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Tonio
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Tonio
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Tonio
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Tonio
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Tonio
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240
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96.0
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38.4
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15.4
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| F
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Tonio
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Tonio
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Tonio
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Tonio
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Tonio
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Tonio
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Tonio
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Tonio
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240
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96.0
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38.4
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15.4
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| G
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Tonio
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Tonio
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Tonio
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Tonio
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Tonio
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Tonio
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Tonio
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Tonio
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6.1
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2.5
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1.0
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0.0
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| H
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Tonio
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Tonio
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Tonio
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Tonio
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Tonio
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Tonio
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Tonio
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Tonio
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6.1
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2.5
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1.0
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0.0
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Results
Ht31P toxicity
Effect of PKA/Epac stimulation
Compilation
| T.TOETS
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S0 & Ht31
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'
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'
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'
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'
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'
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'
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'
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| S0 |
p = |
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CSE (S0) |
p = |
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CSE (Ht31) |
p = |
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| CTR vs CSE |
0,046 |
<0.05 |
vs CTR (Ht31) |
0,052 |
- |
vs CTR (S0) |
0,020 |
<0.05
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| CTR vs CSE+8p |
0,027 |
<0.05 |
vs CSE+8P (Ht31) |
0,271 |
- |
vs CSE+8P (S0) |
0,146 |
-
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| CTR vs CSE+6Bnz |
0,053 |
- |
vs CSE+6Bnz (Ht31 |
0,231 |
- |
vs CSE+6Bnz (S0) |
0,029 |
<0.05
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| CSE vs CSE+8p |
0,633 |
- |
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| CSE vs CSE+6Bnz |
0,080 |
- |
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| CSE+8p vs CSE+6Bnz |
0,062 |
- |
CSE+8p (S0) |
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CSE+8p (Ht31) |
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| Ht31 |
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vs CTR (Ht31) |
0,032 |
<0.05 |
vs CTR (S0) |
0,027 |
<0.05
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| CTR vs CSE |
0,022 |
<0.05 |
vs CSE+6Bnz (Ht31 |
0,324 |
- |
vs CSE+6Bnz (S0) |
0,038 |
<0.05
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| CTR vs CSE+8p |
0,029 |
<0.05 |
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| CTR vs CSE+6Bnz |
0,079 |
- |
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| CSE vs CSE+8p |
0,868 |
- |
CSE+6Bnz (S0) |
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CSE+6Bnz (Ht31) |
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| CSE vs CSE+6Bnz |
0,058 |
- |
vs CTR (Ht31) |
0,115 |
- |
vs CTR (S0) |
0,060 |
-
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| CSE+8p vs CSE+6Bnz |
0,068 |
- |
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| S0 vs Ht31 |
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| CTR |
0,242 |
- |
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| CSE |
0,298 |
- |
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| CSE+8p |
0,148 |
- |
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| CSE+6Bnz |
0,206 |
- |
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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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<a href="http://openwetware.org/wiki/Image:HTERT_Donor_12_P27_%26_P29_240310.xlss">
<img src="http://openwetware.org/images/f/f8/Owwnotebook_icon.png" alt="" width="50" height="50">
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ELISA
