I made a lipid array on nitrocellulose membrane and am using chemiluminescence to detect whether or not my protein binds to the lipids. It works quite well. Except for my last protein: this protein produces a much higher background and all of the lipid spots are white against that background.
Has anyone ever observed white spots and know what its cause is?
White spots against a high background in a chemiluminescence-based lipid array assay are a known issue and usually indicate substrate depletion or signal oversaturation rather than a true negative result. Most likely causes and possible solutions:
Excessive Protein Binding to Nitrocellulose (Non-Specific Binding)
Your protein may bind non-specifically to the nitrocellulose membrane, increasing the overall background.
Solution:
Increase blocking time or switch to a more effective blocking buffer (e.g., 5% BSA instead of milk, or casein-based blockers).
Reduce protein concentration or optimize incubation time.
Overexposure of Chemiluminescence Substrate
Some proteins with high peroxidase activity (if using HRP-based detection) can rapidly deplete the chemiluminescence substrate, resulting in negative or white spots.
Solution:
Reduce the exposure time.
Use a more stable chemiluminescent substrate (e.g., SuperSignal West Dura or Femto instead of ECL).
Strong Lipid-Protein Interaction Leading to Signal Quenching
If your protein binds lipids with very high affinity, it may cause localized substrate depletion.
Decrease the primary antibody or secondary antibody concentration.
Perform a more thorough wash to remove excess protein.
Membrane Drying or Uneven Substrate Distribution
If the membrane dries out or the substrate is not evenly distributed, signal variations can occur. * Ensure even application of the substrate and keep the membrane moist during detection.
Overactive HRP (Horseradish Peroxidase) Conjugate
If the secondary antibody-HRP is too concentrated, it can lead to rapid substrate depletion. * Dilute the HRP-conjugated secondary antibody.