Western Blot Protocol

Download our SDS-PAGE and Western Blot Protocol

Protocol for SDS-PAGE and Western Blotting

SDS-PAGE

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

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

3. 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, bME or an equivalent must be added at an appropriate concentration. Alternatively, non-reduced protein samples can be prepared in the absence of a reducing agent.

4. Cover the gel with 1X running buffer as instructed by the manufacturer.

5. 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.  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.

2. After electrophoresis, remove the gels from the plastic casings and equilibrate in transfer buffer for 10 minutes.

3. Carefully assemble the transfer cassette according to 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.

               

4. Carefully assemble the transfer cassette according to the  illustration below. Sequentially assemble the layers of the transfer sandwich, gently removing any air bubbles with a roller or pipette.

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

5. Once the transfer is complete, gently disassemble the transfer cassette and move 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 to confirm 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.

6. 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 hour with gentle shaking.

IMMUNOBLOTTING

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). Each antibody    should be optimized as required. Incubate the membrane in the diluted primary antibody for 1 hour at room temperature or overnight at 4°C with gentle agitation. 

Optional: To image the molecular weight markers along  with your protein of interest, you can add 1 µg/mL of the Anti-Blue Marker antibody into your primary antibody solution. This antibody will only bind to the blue dye on each molecular weight marker and will not cross react with your protein lysates.  

Tip: Make sure the membrane is completely submerged in the antibody solution to prevent it from drying out.

2. Wash the membrane in 1X TBST three times for 10 minutes each with agitation.

3. Incubate the membrane in an appropriately diluted secondary antibody solution prepared in the same blocking buffer as the primary antibody. Incubate the membrane for at least 1 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 1 µg/mL of the HRP conjugated Anti-Blue Marker antibody to the secondary antibody solution.

4. Wash the membrane in 1X TBST three times for 10 minutes each with agitation.

5. Prepare the chemiluminescent substrate just before use according to the manufacturer’s instructions.

6. Incubate the membrane in the substrate as instructed by the manufacturer.

7. 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