What are the possible causes and solutions for background issues (high, uneven, or speckled)?

Probable cause

Solutions

Too much secondary antibody causing high background

Follow the manufacturer’s guidelines for diluting the secondary antibody and adjust the dilution based on the specific dye in use.

Membrane contamination causing high background

• Use clean forceps and clean incubation trays or dishes to handle the membrane.
• Make sure you’re using the best blocking buffer for your application to avoid cross-reactivity.
• Use detergents after blocking instead of during blocking steps as some detergents can enhance nonspecific background due to auto-fluorescence.

Membrane drying out causing uneven or blotchy background 

• Keep the whole blot completely covered throughout the incubation period to prevent drying out. 
• Agitate consistently during every step of incubation. 

Speckles and fingerprints smudging the membrane

• Use clean forceps to handle the membrane to prevent fluorescence due to contaminants and particulates on unclean tools 
• Avoid touching the membrane directly
• When using the wet transfer method, clean transfer devices thoroughly to avoid speckles. 
• Rinse incubation trays and dishes with methanol followed by water to remove residual dried dyes from earlier experiments.
• Use ethanol to clean the imager surface before capturing the image to remove all traces of dust, residue, and lint. 

Wrong choice of membrane

Background is impacted by nature of the membrane. Switch over to low-fluorescence PVDF (Polyvinylidene Fluoride) membranes if the current PVDF is causing high background due to autofluorescence 

Nonoptimal wash or diluent solutions

• Use 0.05% Tween 20 detergent to prepare the secondary antibody dilution
• Use a wash buffer with 0.1 – 0.2% dilution
• Increase the frequency or duration of washes  

Overloading the protein marker or ladder causing artifacts

Load smaller quantities of molecular weight marker onto the gel