5.3 Experimental Tips

Experimental Procedures
1. Primary Antibody Incubation
Dilution ratio of primary antibody: For the first use, refer to the dilution ratio specified in the antibody instruction manual. If the antibody shows high sensitivity (resulting in very intense bands) after the first use, the dilution concentration can be appropriately increased (i.e., lower the antibody concentration).
Antibody dilution buffer: Antibodies can be diluted in TBST buffer containing 5% BSA or 5% non-fat milk powder. However, if high background was observed in previous experiments, TBST buffer with 5% non-fat milk powder is recommended for dilution.
Incubation conditions for primary antibody: Incubate overnight at 4°C on a shaker.
2. Secondary Antibody Incubation
Dilution ratio of secondary antibody: For the first use, follow the dilution ratio provided in the antibody instruction manual. If the antibody exhibits high sensitivity (leading to overly intense bands) after initial use, the dilution concentration can be appropriately increased (to reduce the antibody concentration).
Incubation conditions for secondary antibody: Incubate at room temperature on a shaker for 1 hour. Prolonged incubation may easily cause non-specific binding, resulting in 杂 bands (non-specific bands) and high background.
Secondary antibody usage: The secondary antibody is for single use only and should not be recovered or reused.
3. Membrane Washing
Washing buffer: Use TBST buffer for washing.
Washing parameters adjustment: If bands were very faint or excessive non-specific bands were observed in previous experiments, the washing time can be appropriately shortened or extended, and the shaking frequency of the shaker during washing can be adjusted (decreased or increased accordingly).
Key note during washing: Keep the membrane in a moist environment throughout the washing process to prevent membrane drying, which would cause high background.
4. Detection
Use of plastic wrap: Ensure the plastic wrap is laid flat without wrinkles wherever it is used. Wrinkles may lead to uneven distribution of ECL solution. Additionally, check that the plastic wrap is intact (free of small holes) to avoid ECL solution leakage and subsequent uneven reactions.
Membrane treatment after ECL incubation: After ECL incubation, blot the membrane with absorbent paper to remove excess solution. Note: Do not allow the membrane to dry completely. Some weak signals may become undetectable once the membrane is fully dry, and background may also increase.
5. Precautions
Ensure the membrane does not dry out during the entire operation process.
When handling the membrane with forceps, grip the corner of the membrane where no detection signal is expected as much as possible.