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| | [[Western blot]] |
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Revision as of 05:16, 25 March 2010
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 Western blot
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Summary
Materials & Methods
Materials
- Transfer buffer (1 L)
- 700 mL H2O (Cold)
- 100 mL 10x Transfer buffer (Cold)
- 0.25 M TRIS (30.3 g/L)
- 1.92 M Glycin (144.1 g/L)
- 1.0% SDS (10 g/L)
- 200 mL Methanol (Cold)
- MIX and Store @ 4 °C
- TBST buffer (4 L)
- 3.6 L UltraPure H2O
- 400 mL 10x TBS
- for 1 L
- 60.5 g/L TRIS
- 87.6 g/L NaCl
- pH 7.6
- 4 mL Tween20
- Mix
- 1x ELFO buffer
- 10x ELFO buffer
- 30.26 g/L TRIS
- 187.60 g/L Glycin
- 10 g/L SDS
Method
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
- 1:1 ratio ECL solutions (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)
Results
--> PICTURES ANOUK
Conclusion
Related
Related topics
Western blot
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Western blot
