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| |} | | |} |
| | ====Co-immunoprecipitation==== |
| | * resuspend beads prepared [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/153| 15March2010]] |
| | * Prepare for all donors (D9 & D12) |
| | # Only beads (30 μL) |
| | # Beads w. antibody (30 μL) |
| | # Beads w. antibody (30 μL) & lysis buffer (200 μL) |
| | # Beads w. antibody (30 μL) & sample (200 μL) |
| | (possible also beads (30 μL) with only sample (200 μL)) |
| | * Incubate ON @ °C |
| ==Results== | | ==Results== |
| *{{todo| ELISA IL-8}} | | *{{todo| ELISA IL-8}} |
Revision as of 05:35, 16 March 2010
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ELISA & hTERT stimulation
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Summary
- New batch of Ht31P to use
- 16Feb2010
- Splitting cells
Materials & Methods
Materials
Method
- Remove medium form 24-wells plate with ASMC's grown ON in DMEM (S0)
- Rinse twice with warm PBS and remove buffer
- Add 200 μL DMEM S0 with either HT31 or HT31P (see schedule below)
- Add 400 μL DMEM S0 to CTR (lane A)
- Add 200 μL DMEM S0 to CSE (lane B)
- Add 200 μL 8-pctp or 6-benz according to schedule below (lane C, D)
- Wait 25 min.
- Prepare 25 mL DMEM S0 with 100% CSE
- Dilute 100% CSE to 45% CSE
- Add 200 μL 45% CSE to wells according to schedule below (lane B, C D)
- Incubate 24 h.
Stimulation set-up
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HT31P 1
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HT31P 2
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HT31P 3
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HT31 4
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HT31 5
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HT31 6
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A (CTR)
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B (CSE)
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C (CSE+8-pcpt)
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D (CSE+6-Benz)
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CTR = control, CSE = Cigarette Smoke extract, 8-pcpt = 8-pcpt-2'-o-me-camp
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 @ 540 nm
- Check duplo standard curve
- Check if values fall within standard curve (preferred)
96-wells plate ELISA
#
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01 S0
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02 S0
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03 S0
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04 Ht31
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05 Ht31
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06 Ht31
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07 S0
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08 S0
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09 S0
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10 Ht31
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11 Ht31
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12 Ht31
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A CTR
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D12 (5March2010)
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D12, P25 (10March2010)
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B CSE
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C CSE+8P
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D CSE+6Bnz
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E CTR
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D9, P21 (11March2010)
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STND 240 pg/mL
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STND 96 pg/mL
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STND 38.4 pg/mL
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STND 15.4 pg/mL
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STND 6.1 pg/mL
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STND 2.5 pg/mL
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F CSE
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STND 240 pg/mL
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STND 96 pg/mL
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STND 38.4 pg/mL
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STND 15.4 pg/mL
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STND 6.1 pg/mL
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STND 2.5 pg/mL
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G CSE+8P
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STND 1 pg/mL
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STND 0 pg/mL
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H CSE+6Bnz
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STND 1 pg/mL
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STND 0 pg/mL
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BLANCO?
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BLANCO?
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Co-immunoprecipitation
- resuspend beads prepared 15March2010
- Prepare for all donors (D9 & D12)
- Only beads (30 μL)
- Beads w. antibody (30 μL)
- Beads w. antibody (30 μL) & lysis buffer (200 μL)
- Beads w. antibody (30 μL) & sample (200 μL)
(possible also beads (30 μL) with only sample (200 μL))
Results
Conclusion
Related
Same actions
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