6 Steps to RUNNING a Successful Western Blot

This is a complete guide to equipment, supplies, and reagents needed for successful Western blot analysis.

Keep reading for an in-depth overview of the Western blotting steps. We’ve also added links to the tools so you can quickly find exactly what you need to complete your experiments.

Multiplex fluorescent Western blot
chemiluminescent western blot
Azure Aqua during Western blotting experiment

Step 1: Gel Electrophoresis

For most experiments, the first step to Western blot analysis is separating the proteins in a sample using polyacrylamide gel electrophoresis (PAGE). In SDS-PAGE (where the proteins in the sample are coated with the detergent SDS), proteins migrate according to their size, with smaller proteins migrating more quickly through the gel. The percentage of polyacrylamide in the gel determines how easily proteins of various sizes can move through the gel, with higher percentage gels having a tighter gel matrix better for resolving smaller proteins. Polyacrylamide gels may be purchased ready-to-use in a variety of percentages or gradients, or gels may be handcast to achieve a customized percentage.

  • Azure Aqua Quad Mini-Cell: a vertical gel running system to carry out electrophoretic separation using 10 cm x 8 cm gels. This also provides everything needed to prepare handcast gels.
  • Azure Aqua Power Supply: can power up to four different Mini-Cell modules simultaneously and users can create and save protocols for electrophoresis and blotting on the power supply.

Step 2: Membrane Transfer

Once protein separation is complete, the proteins are transferred from the polyacrylamide gel to a solid membrane support. Membranes are usually made from nitrocellulose or polyvinylidene difluoride (PVDF). Transfer involves assembling a transfer “sandwich” in which the gel is placed next to the membrane and both are then placed inside a cassette with blotting paper and sponges on either side to ensure a secure fit within the cartridge. Transfer occurs with the sandwich submerged in transfer buffer in a tank, and a current is passed through the sandwich to drive the proteins from the gel to the membrane.

Wet transfer setup for Western blotting
Figure 1. Wet transfer setup. A “stack” is built in which the gel is placed next to a membrane (nitrocellulose or PVDF), both of which have been pre-equilibrated in transfer buffer. Blotting papers and sponge, which have also been pre-soaked in transfer buffer, are added to the outside of the stack so the stack can be held firmly within a cassette that is suspended in the transfer buffer–filled tank. Electrodes on the cassette allow an electric current to be run through the stack so the proteins migrate from the gel to the membrane.

What you need:

  • Azure Aqua Transfer Cell: a tank system to carry out two transfers from minigels to membranes. The Aqua Transfer Cell includes two cassettes, cooling units, and everything necessary for transfer. Cassettes and electrodes are color-coded so you can be sure you set up the transfer with the correct directionality.
  • Azure Aqua Power Supply: powers up to four different modules simultaneously and can create and save protocols for electrophoresis and blotting.
  • Pre-cut Blotting Paper: for transfer sandwiches are available in three sizes, 9 cm x 7 cm, 10 cm x 15 cm, and 13 cm x 18 cm.
  • Pre-cut PVDF Membranes are available in three sizes, 9 cm x 7 cm, 10 cm x 15 cm, and 13 cm x 18 cm.
  • Pre-cut Nitrocellulose Membranes: available in 10 cm x 15 cm and in two pore sizes.
  • Azure Transfer Buffer: formulated for enhanced protein transfer for improved sensitivity.

Step 3: Blocking

Before the target protein(s) can be detected on the blot, non-specific binding sites on the membrane must be blocked by incubating the membrane in a blocking buffer. Home-made blocking buffers contain proteins such as dry milk or serum albumin to block non-specific protein-binding sites.

What you need:

  • Azure Protein-free Blot Blocking Buffer: a protein-free blocking solution optimized for fluorescent Western blots but also appropriate for chemiluminescent detection. This blocking buffer is a good choice for experiments using primary antibodies that cross-react with proteins in home-made blocking buffers.
  • Azure Chemi Blot Blocking Buffer: formulated to enhance signal strength and reduce background in chemiluminescent Western blots.
  • Azure Fluorescent Blot Blocking Buffer: optimized for use with Azure’s fluorescent Western blotting systems and formulated to stabilize fluorescent signal and reduce background noise.

Step 4: Primary Antibody Incubation

The blocked membrane is incubated with an antibody that binds to the target protein of interest. Incubation conditions depend on the antigen-antibody pair. The primary antibody may be diluted in blocking buffer. Learn more about primary antibodies at BosterbioExcess unbound primary antibody is washed away in a series of washes.

Pouring liquid inside incubation tray during Western blotting

What you need:

  • Incubation Trays: available in a variety of sizes that are perfect for washes; trays have lids to prevent dust or other contaminants from contacting the blot
  • Blot Washing Buffer: compatible with all chemiluminescent and fluorescent Western blots
  • Fluorescent Blot Washing Buffer: the best choice for near-infrared fluorescent Western blots Azure’s blot washing buffer is optimized to produce high signal-to-noise ratios with all fluorescent blots.

Step 5: Secondary Antibody Incubation

The presence of primary antibodies bound to their target protein on the blot is detected by binding a labeled secondary antibody to the primary antibody. Secondary antibodies are usually labeled with a fluorophore that can be detected directly, or bound to an enzyme like horseradish peroxidase (HRP) that reacts with a substrate to produce light (chemiluminescence) or a colored product that can be detected visually or on film.

What you need:

Wash Away Excess Secondary Antibody

Finally, the bound secondary antibodies are detected. For chemiluminescent detection, the blot is incubated with a chemiluminescent substrate and the emitted light detected using film or a digital imager. For fluorescent detection, the blot is imaged using a digital imager that has a light source to excite the fluorophore and has the correct filters to detect the emitted fluorescence.

What you need:

  • Chemiluminescent HRP substrate
    • Radiance ECL: provides long-lasting signal and picogram sensitivity
    • Radiance Plus: provides low-femtogram sensitivity for detection of low-abundance proteins
    • Radiance Q: suitable for picogram sensitivity, has a large linear dynamic range developed for CCD imaging
  • Chemiluminescent pen: the Azure ChemWriter ECL is a chemiluminescent pen for annotating chemiluminescent blots.
  • Blot Development Folders: transparent plastic sheets to hold chemiluminescent blots during imaging, a wrinkle-free alternative to plastic wrap.
  • Background Quenching Sheets: placed below the blot when imaging either chemiluminescent or fluorescent blots to reduce background and improve image quality
  • Azure Imaging Systems are CCD systems that can detect chemiluminescence, visible fluorescence, and near-infrared fluorescence; a variety of configurations are available to suit any lab’s imaging needs.
  • The Azure Sapphire Biomolecular Imager is a laser-scanning system capable of imaging chemiluminescence and visible and NIR fluorescence; a variety of configurations are available to suit any lab’s imaging needs.

Step 6: Blot Analysis​

Digital blot images can be analyzed using analysis software, such as AzureSpot Pro. To obtain quantitative information from a Western blot, the signal for the protein of interest can be compared to the signal for a housekeeping protein or to total protein in a process known as total protein normalization (TPN).

What you need:

  • AzureRed Fluorescent Total Protein Stain is a total protein stain for gels and blots that can be used for TPN, can be detected with laser- and CCD-based imaging systems, and is fully compatible with downstream Western blotting or mass spectrometry.
  • TotalStain Q is a fluorescent total protein stain that can be used for TPN. Versions for use with nitrocellulose and with PVDF membranes are available.
  • AzureSpot Pro software facilitates blot analysis, including background subtraction, band detection, and normalization.

READ MORE: What is Total Protein Normalization?

EXPLORE: Azure Imaging Systems, Azure Sapphire Biomolecular Imager

AzureSpot Pro analysis Software


New to Western blotting? Need to troubleshoot your Western blot?​ Want to brush up on Western blotting best practices? Claim your free Western Blotting eBook!

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Two scientists looking at screen on Azure 600 Western blot imager
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