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Most adherent mammalian cell lines are passaged when 70–90% confluent. At standard density, passage every 2–4 days (monitoring daily). Suspension cells are split based on cell count and viability, typically every 2–3 days. Primary cells may require more frequent monitoring.
Primary antibody: 1–2 hours at room temperature or overnight at 4°C. Secondary antibody (HRP/AP conjugated): 1 hour at room temperature. Blocking: 1–2 hours. Substrate development: 10–30 minutes (stop when colour is visually optimal or at maximum linear range). TMB substrate is typically stopped at 30 minutes.
E. coli at 37°C: visible colonies in 12–18 hours. Overnight incubation (16–18 hours) is standard for most gram-negative bacteria. Slow-growing organisms (Mycobacterium): days to weeks. Anaerobes require oxygen-free conditions for incubation and may take 24–48 hours for visible growth.
Overcrowding (above 90% confluence) causes contact inhibition in non-transformed cells, nutrient depletion, pH drop (acidification), and metabolic waste accumulation. Transformed cell lines may continue growing but produce unhealthy cells with poor experimental consistency.
Room temperature with gentle rocking: 1–2 hours (standard). 4°C overnight: preferred for better signal-to-noise ratio (antibody has more time to bind at lower off-rate). Overnight at 4°C consistently produces cleaner blots with less background compared to shorter room-temperature incubations.