Western Blot

Transfer (semi-dry)

• transfer gel into Blotting Buffer (BB)
• cut six pieces 3MM blotting paper and a PVDF membrane to size of gel
• soak three blotting paper in BB, lay on anode roll over each paper with 25 ml pipette to remove air bubbles
• wet PVDF membrane in ethanol, soak in BB, lay on blotting paper, roll
• lay gel on PVDF membrane,
• lay three more blotting paper on top, roll with pipette
• apply 0.8 mA per cm² for 1.5 h
• air dry membrane

Detection

• wet membrane in ethanol, wash in destilled water
• stain with Poinceau-S, wash in destilled water
• wash in Blocking Buffer 30 min
• expose to primary antibody diluted in Blocking Buffer 1-2 h. For reuse, keep antibody with NaN₃ at 4ºC
• wash 4x ~5 min in Blocking Buffer
• expose to secondary antibody diluted in Blocking Buffer 1 h
• wash 4x ~5 min in Washing Buffer (1x PBS or TBS, 0.05% TritonX-100)

for alkaline phosphatse conjugates:

If PBS was used, wash 2x in 50 mM Tris buffer. Wash in water, then expose to NBT/BCIP substrate (e.g. WesternBlue Stabilized Substrate for Alkaline Phosphatase, Promega). Color develops in ~10 min.

for peroxidase (HRP) conjugates:

use Amerham ECL detection or chloronaphtol-substrate (less sensitive):

• dilute 6 mg 4-chloro-1-naphtol in 2 ml methanol or ethanol
• add 10 ml PBS
• add 10 µl 30% H₂O₂

Color develops slowly.

Stripping

wash 1 h in

• 0.2 M glycine, pH 2.2 (HCl)
• 0.1% SDS
• 1% Tween 20