Replace the blocking solution Many commonly used blocking solutions may mask epitopes on targets, making them difficult to detect. Optimization 10ul Pipette tips conditions and increasing stripping time need to ensure that the blotting membrane is completely submerged in the stripping buffer and shaken fully on a shaker. However, if you are already accustomed to using chemiluminescence Western blots, your markers may be loaded too much. The loading buffer is not fresh or contains too much salt. In addition, increasing the amount of rinsing agent or using a stronger rinsing agent, such as SDS or NP-40,
can further reduce the background. High background caused by high primary antibody concentration 4. Insufficient rinsing Solution: 1. Increase the volume of the primary antibody incubation solution and verify that the solution completely covers the membrane during the incubation step. 2. Many commonly used blocking solutions may mask epitopes on targets, making them difficult to detect. Insufficient contact between the primary antibody and the blot. Incomplete blocking, non-specific binding to unrelated lysing proteins. It has many technical links and long operation time. Running the gel too fast, causing the gel to heat up and degrade the polymer.
Insufficient blocking 2. Question: The band is wavy, smiling, and skewed. Purify the primary antibody or replace the antibody 3. Increase the dilution of the primary antibody or increase the incubation time. Problem: The high background of the fluorescent western blot is caused by: 1. Increase the dilution of the primary antibody or increase the incubation time. Incomplete blocking, non-specific binding to unrelated lysing proteins. When assembling the sandwich,
pay attention to completely remove the air between the blotting membrane and the gel. Incubation at 4 degrees can reduce the non-specific binding problem: The stripped blotting membrane still has a band Cause: Incomplete stripping Solution: 1. It may be necessary to replace the sponge pads to achieve a suitable tightness. Problem: Wave-shaped surrounding strips Cause:
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