Anisha Patel COSMOS UC Davis Cluster 1: Biotechnology

PART 228. Soak the filter pads in PVDF Transfer Buffer.29. Assemble the membrane sandwich and place in transfer assembly.30. Fill the transfer tank with PVDF transfer buffer.31. Remove the cooling block from storage at 20 degrees Celsius; place into transferapparatus.32. Place the tank in a refrigerator at 4 degrees Celsius.33. Block the membrane in 5% Non Fat Dry Milk (NFDM) in TBS with 0.05% tween-20for 2 hours.34. Incubate the membrane with the primary antibody for 16 hours at 4 degrees C. Dilutethe antibody with a 2.5% Non Fat Dry Milk (NFDM) in Tween TBS solution. A totalantibody and diluent solution of 5 ml will coat a small membrane in a rotating tube verywell.35. Remove the antibody and perform 3 room temperature washes (10 minutes of rotationeach) with Tween TBS.36. Reblock the membrane in 10% Non Fat Dry Milk (NFDM) in Tween TBS for 10minutes at room temperature.37. Incubate the membrane with the secondary antibody for 30 minutes at roomtemperature. Dilute the antibodywith a 2.5% 38. Non Fat Dry Milk (NFDM) in Tween TBS solution.39. Remove the antibody and perform 3 room temperature washes (10 minutes of rotationeach) with Tween TBS.