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Western Blotting w/Tween 20 Blocking

Tween-20 can be used, in lieu of proteins like BSA or milk, to block both nitrocellulose and PVDF membranes for western blotting
Starting after transfer
Optional: Dry blot to potentially help ‘fix’ proteins
Note: PVDF will likely need re-wetting with 100% methanol if you do this

  • Blocking: TBST w/0.5% Tween 20 for 30min
    • (Supplement TBST with 1/100th vol of 25% Tween 20 to get to 0.5%)
    • This website says you can block in only a few min if you use 2% Tween 20
  • 1° = Dilute in TBST, incubate 30-60min at RT
  • Wash 3x 5min w/TBST or 1x 10min
  • 2° = Diute in TBST, incubate 30-60min at RT
  • Wash 3x 5min w/TBST
  • Develop w/whatever

Source for Tween concentrations:
You may get much higher signal from this, which would require a more dilute primary and/or secondary antibody.


10mM Tris; pH 8 1.21 g/L
150mM NaCl 8.77 g/L
0.25% Tween 20 2.5 g/L