Q1: Weak stripe signal or no band?
A1:
1) The protein content of interest in the sample is low. Increase the amount of loading appropriately;
2) The protein of interest is not completely transferred to the membrane. Optimize the transfer conditions, recommend the buffer replacement, ensure that the membrane is pretreated before use, and determine that there are no air bubbles between the glue and the membrane during the transfer;
3) The antibody use concentration is low or the incubation time is not enough. Increase the antibody use concentration or incubation time;
4) The exposure time is too short. Appropriately extend the exposure time;
5) Local substrate consumption is too fast. Reduce the amount of loading, optimize the concentration of antibodies used, and increase the amount of ECL substrate.
Q2: Is the background too high?
A2:
1) Insufficient closure. Optimize the type, concentration and sealing time of the blocking agent;
2) The washing film is not sufficient. Increase the time and frequency of film washing;
3) The antibody concentration is too high. It is recommended to optimize according to the recommended dilution ratio of the antibody supplier, and the optimal use of the antibody can be explored for gradient dilution;
4) Overexposure. Reduce exposure time.
