Western Blot Protocol

Posted on Dec 8, 2014

The following is AxioMx’s recommended western blot protocol. This protocol is applicable when primary antibodies are either FLAG®-labeled scFvs or IgGs.

A. Buffers

Lysis buffers

Buffers may be stored at 4°C for several weeks or for up to a year aliquoted and stored at -20°C.

Nonidet-P40 (NP40) buffer

150 mM NaCl

1.0% NP-40 (possible to substitute with 0.1% Triton X-100)
50 mM Tris-HCl pH 8.0
Protease Inhibitors

Commercially available Cell lysis buffers

B-PER® Complete Bacterial Protein Extraction Reagent (Pierce cat # 89822)

BugBuster® Extraction buffer (EMD cat # 70921-5)

Laemmli 2X buffer / loading buffer

4% SDS

10% 2-mercaptoethanol

200 mM DTT (optional)

20% glycerol

0.004% bromophenol blue

0.125 M Tris-HCl

Check the pH and adjust pH to 6.8.

Running buffer (Tris-Glycine/SDS)

25 mM Tris base

190 mM glycine

0.1% SDS

Check the pH, which should be about pH 8.3. Adjust if necessary.

Transfer buffer (Wet)

25 mM Tris base

190 mM glycine

20% methanol

Check the pH, which should be about pH 8.3. Adjust if necessary.

Blocking buffer:

5% milk to PBST buffer.

B. Protocol:

  1. Load desired amount of protein into the wells of the SDS-PAGE gel, along with molecular weight markers.  Load 10-100 ng of total purified target protein.
  2. Run the gel(s) at 200V for 1 hour.
  3. Transfer the proteins in the gel to a nitrocellulose membrane using the BioRad Trans-Blot® Turbo™ Transfer System. See (http://www.bio-rad.com/webroot/web/pdf/lsr/literature/10020688.pdf) for operation instructions.
    1. Alternatively transfer using wet or semi-dry transfer.
  4. Block membranes for 1 hour at room temperature on a platform shaker. Alternative: block membranes at 4ºC overnight (covered) on platform shaker if western blots are not performed the same day
  5. Incubate membranes with appropriate dilutions of primary antibody in 5 % MPBST for 1 hour at room temperature on a platform shaker.
    1. Alternatively incubate overnight at 4°C.
  6. Wash the membranes three washes in PBST, 5 minutes each.
  7. Incubate the membrane with the recommended dilution of the secondary in 5% MPBST for 1 hour at room temperature on a platform shaker.
    1. Please note:  2º antibody for AxioMx scFv (anti-FLAG-HRP) at suggested dilution in 5% MPBST.
  8. Wash the membranes three washes in PBST, 5 minutes each, then rinse with PBS.
  9. For signal development, develop blots with ECL Reagent.
    1. Mix together equal volumes of the Clarity™ Western ECL Substrates (BioRad Clarity® Western ECL Substrate (Cat. #170-5061, 500mL)).  Follow the kit manufacturer’s recommendations.
  10. Remove excess reagent.  Cover blot with plastic wrap while awaiting imaging.
  11. Image blot using development techniques for chemiluminescence or normal imaging methods for colorimetric detection.

FLAG® is a registered trademark of Sigma-Aldridge Co. LLC

Transblot® is a registered trademark of BioRad Laboratories, Inc.

B-PER® is a registered trademark of Thermo Fisher Scientific Inc.

BugBuster® is a registered trademark of EMD Millipore, a division of Merck KgaA.

Click Here to Download the AxioMx Standard Western Blot Protocol