Western blot: no bands

1. Ranked causes

   1. Primary antibody inactive, wrong host, or wrong dilution (lot / storage)Loading-control antibody and Ponceau still show protein; target antibody gives blank lanes.
   2. Transfer failed — protein never reached the membranePre-stained ladder absent from membrane; gel retains dye after transfer.
   3. Detection substrate / secondary conjugate expired or mismatched speciesFresh ECL + species-matched secondary recovers bands on a control lysate.
   4. Blocking or wash conditions strip the epitope before detectionShortening block time or switching milk→BSA restores signal on a known positive.
   5. Imager exposure / gain set below detection (or wrong channel)Same membrane shows bands after longer exposure or on a second imager.

2. Recovery steps

   1. Confirm transfer with Ponceau/ladder on the membrane before blottingSeparates transfer failure from antibody/detection failure in one glance.
   2. Re-probe with a fresh primary aliquot at a validated dilution (e.g. 1:1000)Rules out inactive or mis-stored primary antibody.
   3. Switch blocker to 5% BSA if the target is phospho-sensitive to milk caseinMilk can mask phospho-epitopes; BSA is the usual alternative.
   4. Increase imager exposure stepwise (30 s → 2–5 min) on a positive-control laneUnder-exposure is a common silent failure after a camera preset change.

3. Controls first

   • Did the positive-control lysate / known-good sample also show no bands?If yes: Transfer, antibody, or detection chemistry — not this sample alone.
   • Is the pre-stained ladder visible on the membrane after transfer?If no: Fix transfer (time/voltage/buffer/stack) before troubleshooting antibodies.

Diagnose your run

Opens Diagnostics capture with this first-party playbook behind it. Sign in if the workspace asks.