Sample Preparation
|
- Proceed the routine SDS-PAGE, transfer membrane and Western Blot steps. 2. Blot membrane: wash membrane with TBS-T twice, 10 min each. Immerse membrane into 5% defatted milk blocking solution, shake by a shaker, and incubate at room temperature for 1 hour. 3. Remove the blocking solution, incubate the membrane in properly diluted primary antibody at room temperature for 2 hours, or at 4℃ overnight on a shaker. Note: Dilute the primary antibody with blocking solution at the antibody manufacturer's recommended dilution. 4. Wash the membrane with 4× or 6× TBS-T for 3 times, 10 min each. Increase the volume of wash buffer, and / or the wash times can reduce the background. Note: Before proceeding Step 4, rinse the membrane with TBS-T simply, the wash efficiency will be increased. 5. Incubate the membrane in properly diluted HRP conjugated secondary antibody at room temperature for 2 hours on a shaker. Note: Dilute the secondary antibody at the antibody manufacturer's recommended dilution. 6. Repeat Step 4 to remove the non-specific conjugation of HRP conjugated secondary antibody. Chongqing Biospes Co., Ltd Product Manual
|