How do I optimize chemiluminescent western blots?

Chemiluminescent Western blots are used for their high sensitivity and ability to detect very low levels of protein. However, they can be difficult to perform due to high background noise and difficulty in obtaining a clear signal from the protein of interest. Here are some tips to optimize chemiluminescent Western blots:

• Clean all equipment and trays thoroughly, handle gel and membranes with gloves, and cover trays during incubation to avoid contamination and prevent background.
• Optimize protein loading onto the gel. Generally, you can load 20-40 μg total protein without overloading the well, but this should be adjusted for each protein-antibody pair to avoid overloading or signal saturation.
• Mix buffers thoroughly and filter before using to prevent high background from particulates in buffers sticking to membranes.  
• Choose the correct membrane. Both, PVDF (Polyvinylidene Fluoride) and nitrocellulose membranes, which are widely used, have unique benefits and should be chosen based on your experiment’s needs.
• Use a checkerboard and dot blot titration to find the ideal concentrations of primary and secondary antibodies for clear signal detection.
• Experiment with different blocking buffers to prevent interference with detection caused by blocking buffers masking the protein of interest or antibodies reacting with proteins in blocking buffers. 
• When using buffers containing sodium azide, do not dilute horseradish peroxidase (HRP)-conjugated secondary antibodies, as sodium azide impedes HRP activity.
• Experiment with increasing the washing buffer volume and the frequency and duration of washes if you’re experiencing high background issues. 
• To boost sensitivity and signal duration, experiment with different substrates that are suited for high or low abundance proteins and have varying reaction rates and light emission durations.
• Equilibrate the substrate to room temperature before using to enhance enzyme activity.
• Use sufficient substrate to coat the blot completely to prevent variations in local concentrations and signal intensity. 
• Opt for a digital imager over film to increase the linear dynamic range, which makes it easier to detect low-abundance proteins without oversaturating high-abundance signals.