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 Co-immunoprecipitation part III
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Summary
The samples have been incubated with the (PKA) antibody coated beads and today the beads are washed and stored in Laemli buffer
Materials & Methods
Materials
- Laemli buffer
- PBS
- syringe
- Hot lysis buffer
Method
Co-immunoprecipitation
- Centrifuge sample incubated with beads, 5 min. 14.000 RPM
- Remove supernatant* (w. syringe)
- Add 1 mL PBS
- Centrifuge, 5 min. 14.000 RPM
- Remove supernatant*
- Add 75 μL Laemli buffer to beads
- Store samples @ -20 °C
- Alternatively go directly on to Western blot (Boil samples and spin beads down, bring supernatant onto gel)
* Be careful not to touch beads
- Use cells grown by Bachelor students TODO
- Remove medium
- Wash with PBS twice and remove
- Add 600 μL S0 or S0+Ht31 (see hTERT VASP schedule below)
- Incubate 15-20 min. RT
- Add 600 μL S0 or S0+6-Bnz (see hTERT VASP schedule below)
- Incubate 10 min. RT
- Add 600 μL S0 or S0 45% CSE (see hTERT VASP schedule below)
- Incubate 10 min. RT
- Remove medium
- Wash with PBS and remove
- Add 200 μL Hot Lysis buffer and scrape cells with tip
- Transfer cell lysate into 1.5 mL tubes
- Store @ -20 °C
hTERT VASP
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1
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2
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3
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| A
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Control
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Ht31 (50 μM)
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CSE (15%)
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| B
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CSE (15%) + Ht31 (50 μM)
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CSE (15%) + 6-Bnz (500 μM)
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CSE (15%) + 6-Bnz (500 μM)+ Ht31 (50 μM)
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Coating of plate
- Prepare coating buffer
- 1.24 g Na2CO3 in 100 mL (Buffer A)
- 2.52 g NaHCO3 in 300 mL (Buffer B)
- Take 70 mL of Buffer A
- Add Buffer B until pH of 9.6 has reached (150 - 200 mL)
- Continue as described 18Feb2010
BSc.
- Alamar blue
- Stop stimulation (hTERT)
- Cells to 96-wells (hTERT)
Results
Related entries
Run 2: Ht31 Dose/Effect Curve & S0 D9/D12
Same action
Related topics
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Co-immunoprecipitation part III
