Western Blot  (NEB #E8033)

For monoclonal antibodies: #E8032 Anti-MBP, #E8033 Anti-M13 pIII, #E8034 Anti-CBD and #E8038 Anti-MBP (HRP Conjugated)

For polyclonal antibodies: #E8023 Anti-GLuc and #P9310 Anti-SNAP-tag

Modifications: For Anti-GLuc blots use 2.5% BSA-and- 2.5% non-fat milk in TBST.  For Anti-MBP(HRP-Conjugated) a secondary antibody is not necessary.

Protocol
  1. Block membrane in 25 ml of TBST + 5 % nonfat milk (or desired blocking buffer) for 1 hour at room temperature or overnight at 4 °C with gentle agitation.
  2. Wash membrane 3 times for 5 minutes each with 15 ml of TBST Wash Buffer.
  3. In 20ml of Blocking Buffer with gentle rocking overnight at 4ºC or for 1 hour at room temperature, incubate membrane with primary antibody at a 1:1000 dilution except for anti-MBP use 1:10,000 dilution and for anti-MBP (HRP conjugated) use 1:2000-5000.
  4. Wash membrane 3 times for 5 minutes each with 15ml of TBST Wash Buffer.
  5. In 20 ml of Blocking Buffer, incubate the membrane with a commercially available secondary antibody, with gentle rocking for 1 hour at room temperature. A secondary antibody is unnecessary for detecting anti-M13 MBP (HRP-conjugated). Proceed to step 6.
  6. Wash membrane 3 times for 5 minutes each with 15 ml of TBST Wash Buffer.
  7. Proceed to chemiluminsecent detection of protein bands with a commercially available detection reagent.

TBS: 20 mM Tris-HCl (pH 7.5), 150 mM NaCl. (may be made as a 10x stock, autoclaved and stored at room temperature)

TBST : 1xTBS + 0.1% Tween-20