Western Blot Protocol

Protocol for SDS-PAGE and Western Blotting


  1. Prepare or purchase an appropriate percentage polyacrylamide gel to best resolve your protein of interest based on its estimated molecular weight.

Tip:Low molecular weight proteins are best resolved on high percentage gels (15-20%), whereas large proteins 200+ kDa will require 4-6% gels for sufficient resolution. If your protein of interest has multiple isoforms of low to high molecular sizes, gradient gels would be your best option for achieving efficient separation of proteins.

Precast gels with different formats and prepared electrophoresis buffer stocks are available from a number of manufacturers which make SDS-PAGE more reproducible and convenient.

  1. In the first well, load a protein molecular weight standard.  Novus scientists typically run a pre-stained standard to monitor the progress of the electrophoresis. Optional:  If you wish to visualize the molecular weight markers on X-ray film or on a digital photograph, we recommend running 1-4 µL of an all blue prestained molecular weight marker and use the mouse monoclonal Anti-Blue Marker Antibody (6F4-F6) (NBP2-33376) in conjunction with your primary antibody.  If HRP is the method of detection for your Western blot a convenient Anti-Blue Marker Antibody (6F4-F6) (NBP2-33376H) - HRP conjugate is also available.
  2. Load protein lysates (10-50 µg) prepared in 1X Laemmli sample buffer into the remaining wells of the gel.

Tip: For preparing reduced protein samples, a reducing agent such as DTT, ϐME or an equivalent must be added to an appropriate concentration.Alternatively, non-reduced protein samples can be prepared in the absence of a reducing agent.

  1. Cover the gel with 1X running buffer as instructed by the manufacturer.
  2. Run the gel as recommended by the manufacturer.  Voltage may vary depending on research needs.


Protein Transfer

  1. Prepare all membranes, filter papers and buffers before starting the protein transfer procedure.

Tip:For proteins with a molecular weight less than 30 kDa, use 0.2 µm PVDF membrane, otherwise 0.45 µm PVDF is recommended.Briefly, wet the PVDF membrane in methanol and then soak it in distilled water followed by transfer buffer.Handle the PVDF membrane carefully to avoid marring or scratching the surface as this can increase background during immunoblotting.

  1. After electrophoresis, remove the gels from the plastic casings and equilibrate in transfer buffer for 10 minutes.
  2. Carefully assemble the transfer cassette according the illustration below.  Sequentially assemble the layers of the transfer sandwich, gently removing any air bubbles with a roller or pipette.

Tip:Make sure no air bubbles are trapped between the gel and the membrane as they will impede the transfer of protein to the membrane.


                                     Diagram of a typical wet protein transfer sandwich


  1. Perform the transfer of proteins from the gel to the membrane according to the manufacturers’ recommendations.

Tip:Low molecular weight proteins <30 kDa require a short transfer time to avoid pulling the protein through the membrane.

  1. Once the transfer is complete, gently disassemble the transfer cassette and remove the membrane to a clean surface to mark the molecular weight bands with a China marker or pencil.  If all blue molecular weight markers have been used, there is no need to mark the bands as they will be visible on your X-ray film or digital image when used in conjugation with the Anti-Blue Marker antibody.

Tip: At this point the membrane can be stained with a reversible protein stain like 0.1% Ponceau S for 1 or 2 minutes for confirming a successful protein transfer.Rinsing the membrane in distilled water will remove the excess stain and reveal the protein in each lane.Additional marking of the lanes is possible at this time.

  1. Remove the excess stain by soaking the membrane in 1X TBST and then transfer to a blocking solution of choice.  Novus Scientists typically block their membranes in 5% non-fat milk in TBST or in 5% BSA in TBST for 1 h with gentle shaking.

Tip:Block membrane in 5% BSA if the antibody for Western Blot analysis is targeted to phospho-epitopes, as milk block contains many phosphoproteins which can interfere with detection.

Optional:  The membrane can be air dried at this point and stored for later use.  Rehydrate the membrane by soaking in 50% methanol fallowed by several washes in TBST.



  1. Prepare a working dilution of the primary antibody in 1% non-fat milk -TBST or 1% BSA -TBST.  Typical starting points for dilutions are 1-2 µg/mL (check the product datasheet), however, each antibody should be optimized as required.  Incubate the membrane in the diluted primary antibody for 1 hour at room temperature to overnight at 4°C with gentle agitation.  Optional:  To directly image the molecular weight markers along with your protein of interest, you can add the Anti-Blue Marker antibody into your primary antibody solution to 1 µg/mL concentration.  This antibody will only bind to the blue dye on each molecular weight marker and will not interfere or cross react with your protein lysates.  

Tip:Make sure the membrane is completely submerged in the antibody solution to prevent it from drying out.The primary antibody dilution can be stored for later use at 4⁰C with the addition of sodium azide to 0.05%.

  1. Wash the membrane in 1X TBST three times for 10 minutes each with agitation.
  2. Incubate the membrane in an appropriately diluted secondary antibody solution prepared in the same blocking buffer as the primary antibody.  Incubate the membrane for 1-2 hour at room temperature.  Optional:  To detect the molecular weight markers at the same time as your protein of interest, add an anti-mouse HRP conjugated antibody into the secondary antibody solution (if not already anti-mouse) or add the HRP conjugated Anti-Blue Marker antibody to the secondary antibody solution to 1 µg/mL concentration.
  3. Wash the membrane in 1X TBST three times for 10 minutes each with agitation.
  4. Prepare the chemiluminescent substrate just before use according to the manufactures instructions.
  5. Incubate the membrane in the substrate as instructed by the manufacturer.
  6. Sandwich the membrane between layers of plastic (ex. sheet protectors) and expose to X-ray film or digitally capture images.

More Resources:

Western Blot Troubleshooting

Western Blot Video

Western Blot Illustrated Assay