Transfection reagent |
Lipofectamine LTX |
FuGENE HD (E2311, Promega, Madison, WI, USA), and Polyethylenimine HCL MAX (24765, Polysciences, Taiwan) |
Cell lysis buffer |
Using both RIPA and denaturing lysis buffers |
1lysis % NPbuffer 40, RIPA alone buffer alone, and denaturing |
Antigen preparation |
Pellets of lysed cells |
Supernatants of lysed cells, and whole cells lysed in the dish without pelleting |
Time of collection of transfected cells (hours post transfection) |
48 |
60 |
Antigen concentration |
25 μg/well (100 μL/well at 0.25 μg/μL) |
6.25, 12.5, 50, 100, and 200 μg/well |
Blocking reagent |
PBS containing 1% Block ACE |
c-block-e, h-block-e, k-block-e, and b-block-e (BCL-BKSE-01, Beacle, Kyoto, Japan) |
Primary sera/plasma dilution |
1:50 (100 μL/well) diluted in PBS-T containing 0.4% Block ACE |
1:100 |
HRP-conjugated protein A/G dilution |
1:10,000 (100 μL/well) diluted in PBS-T containing 0.4% Block ACE |
1:5,000 |