Over-Developed Plate with Excessive Background Color

Symptom

The entire membrane has excessive background color development, reducing contrast between spots and background. Spots may be obscured by high background signal.

1. 1 Enzyme substrate incubation time exceeding 1 hour
2. 2 Plate not dried properly before reading
3. 3 Insufficient washing steps allowing reagent accumulation
4. 4 Foam formation during washing preventing effective and uniform wash coverage

1. 1 Limit enzyme substrate incubation to maximum 1 hour; monitor development with microscope
2. 2 Dry plate longer before reading, protected from light; overnight drying at 4°C increases contrast
3. 3 Wash both sides of membrane thoroughly with distilled water before and after development
4. 4 Avoid squirt bottles with narrow spouts that produce excessive foam; use proper wash technique

Related Video (2)

Mabtech ★ 85

ELISpot tutorial: step-by-step full assay protocol

"Step-by-step protocol tutorial directly teaches substrate incubation timing and best practices to avoid over-development"

Animated biology With arpan ★ 72

Elispot assay ( assay to determine cytokine producing cells)

"General ELISpot assay overview covers technique fundamentals and substrate application procedures relevant to preventing background artifacts"