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Electroblotting of Protein

Semi-Dry Electrophoretic Blotting of Protein

Transfer Buffer

Bjerrum and Schafer-Nielsen transfer buffer containing SDS:
48 mM Trizma base, 39 mM glycine, 20% methanol, 0.00375% SDS

Dissolve 5.82 g of Trizma base and 2.93 g of glycine in about 700 ml of H2O.
Add 1.875 ml of 20% SDS and 200 ml of methanol.
Adjust the volume to 1 liter with H2O.

Electrophoretic transfer with Bio-Rad Trans-Blot SD.

1. Following electrophoresis, equilibrate the gel in transfer buffer.

2. Cut nitrocellulose membrane to the dimensions of the gel. Wet the membrane in the transfer buffer for 15-30 min.

3. Cut two pieces of filter paper (Whatman 3MM) to the dimensions of the gel. Saturate the filter paper with the transfer buffer.

4. Place a pre-soaked sheet of the filter paper onto the platinum anode of the Trans-Blot SD.

5. Place the pre-wetted blotting media on top of the filter paper.

6. Place the equilibrated gel on top of the transfer membrane, aligning the gel on the center of the membrane.

7. Place another sheet of pre-soaked filter paper on top of the gel.

8. Carefully place the cathode onto the stack. Press to engage the latches with the guide posts without disturbing the filter paper. Place the safty cover on the unit.

9. Transfer mini-gels for 15-30 minutes at 10-15 V. Large gels can be transfered for 30-60 minutes at 15-25 V. Do not exceed 25 V. A current limit (3 mA/cm^2 for large gels; 5.5 mA/cm^2 for mini-gels) is recommended to prevent over-heating.

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