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| ==Notes== | | ==Notes== |
| * No ice block was put in transfer system for the first 24 min. (of 60 min. in total) | | * No ice block was put in transfer system for the first 24 min. (of 60 min. in total) |
| | * Membrane was blocked for 1.5 h @ RT (14.22 - 15.52) |
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| ==Results== | | ==Results== |
| *{{todo| WHAT?}} | | *{{todo| WHAT?}} |
Revision as of 06:41, 22 March 2010
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Summary
- Co-IP Day I
- Western blot Co-IP
- Put cells to S0
Materials & Methods
Materials
Method
Co-immunoprecipitation part I
- Beads were coated with anti-PKA antibodies.
- For 2 samples (D9/D12; Only Beads, Beads with Antibody, Beads w. AB & RIPA and Bead w. AB & Sample)
- 280 μL beads
- Spin down and remove EtOH
- Wash with excess (400 μL) PBS
- Spin down and remove PBS
- Add 1:1 PBS:Beads (280 μL)
- 200 μL for Beads with Antibody, Beads w. AB & RIPA and Bead w. AB & Sample (D9/D12)
- 80 μL for Only Beads (D9/D12)
- (30 μL for each condition)
- Add 1:100 (PKA RIIβ) antibody (or not for control), 2 μL
- Incubate ON @ 4 °C
SDS-PAGE
- Clean the glass plates (UP, Soap, UP, EtOH and dry)
- Prepare elaboration, look out for leakage!
- Choose % of gel, big proteins require higher % (here 10% SDS gel)
- Prepare separating gel (pour until green beam, ~10 mL per gel)
- When polymerized (approx. 30 min) remove isolating layer (wash 3x with UP when isobutanol, remove UP with tissue paper)
- Prepare 5% stacking gel
- Pour until flooding and add combs
- Dilute samples with the buffer which they are stored in until an equal amount is reached (20 ng in 30 μL)
- Add 10 μL 4x loading buffer
- Boil samples for 5 min. and cool samples on ice
- Bring gels into the electrophoresis elaboration and pour ELFO buffer in the inner space to check for leakage
- Remove combs and rinse slots with buffer
- Fill slots with sample (40 μL) and marker (15 μL)
- Start electrophoresis (40 min. 200 V)
- Alternatively start with 100 V until samples reach the separating gel and increase voltage
Western Blot
- Moisten sponges, filter papers and membrane in transfer buffer
- Prepare sandwich
- Black clamp
- Sponge
- Whatman paper
- Gel
- Membrane
- Whatman paper
- Sponge
- White clamp
- Put sandwhich in the transferring system (black to black) and fill it with transferbuffer
- Transfer proteins to membrane for 1 hour @ 100 V on ice while stirring
- After transfer check if marker is visible on membrane (successful transfer)
- Alternatively Ponceau S staining is possible (not used @ this lab)
- Remove LEFT UPPER corner to mark what is what
- Block membrane with 5% milk solution in TBST buffer (ON, 4°C)
- Treat with primary antibody (diluted in 5% TBST-milk solution) (ON, 4°C)
- GAPDH 1:2000
- VASP 1:1000
- AKAP 1:1000
- Wash 3x 10 min. in TBST buffer (first can be less)
- Treat with secondary antibody (diluted in 5% TBST-milk solution) (1h, RT)
- Wash 3x 10 min. in TBST buffer
Preparing film
- 1:50 ratio ECL solutions B:A (2 mL per membrane)
- Incubate membrane in ECL solution 5 min, RT (both sides of membrane)
- Place blot in between two overhead sheets
- Take picture (depends on what is available)
Notes
- No ice block was put in transfer system for the first 24 min. (of 60 min. in total)
- Membrane was blocked for 1.5 h @ RT (14.22 - 15.52)
Results
Conclusion
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Project name
