Western Blot (NEB #E8033)
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
- 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.
- Wash membrane 3 times for 5 minutes each with 15 ml of TBST Wash Buffer.
- 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.
- Wash membrane 3 times for 5 minutes each with 15ml of TBST Wash Buffer.
- 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.
- Wash membrane 3 times for 5 minutes each with 15 ml of TBST Wash Buffer.
- 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