Week 3: Blotting

November 21, 2011

PURPOSE

This week the Western blots were prepared and put on a transfer unit.

METHODS

1. CUt 6 pieces of the whatman paper and 1 piece of the nitrcellulose (NC) paper the exact size of the SDS gel.

2. Mark one corner of the NC paper with a pencil.

3. Float NC paper on transfer buffer then submerge.

4. Soak the whatman paper in transfer buffer.

5. Rinse the gel in dH2O.

6. Construct a "gel sandwich" for transfer using the following order:

POSITIVE TERMINAL

WESTERN APPARATUS GRID

3 SHEETS FILTER PAPER

NITROCELLULOSE PAPER

SDS-PAGE GEL

3 SHEETS FILTER PAPER

WESTERN APPARATUS GRID

NEGATIVE TERMINAL

7. RUn the transfer unit for one hour at the required mA.

8. Disassemble the transfer unit and block NC in 50 mL of Blotto overnight.

9. Wash NC with 20 mL TBS-T, 1x15 min, 2x 5 min (Swanson)

10. Incubate NC in primary antibody solution (TBS-T) until the following week.

RESULTS

Results were not obtained this week.