6 Steps to Western Blotting

The six Western blot steps outlined below serve as the complete guide to everything you need for successful Western blot analysis, including  equipment, supplies, and reagents.

Multiplex fluorescent Western blot

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

Six steps to Western blotting

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, the proteins in the sample are coated with the detergent SDS. The proteins then 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. 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. You can also choose to hand cast your own gels to achieve a customized percentage.

What you'll need for gel electrophoresis:

  • Azure Aqua Quad Mini-Cell: Designed for running 1–4 precast or handcast gels (cassette size 10cm x 8cm). The unit comes equipped with four gel casting stands and casting frames. This is perfect for handcasting your own gels.
Loaded gel in electrophoresis for SDS-PAGE
The Azure Aqua Quad Mini Cell is a vertical gel running system that carries out electrophoretic separation using 10 cm x 8 cm gels. It provides everything needed to prepare hand cast gels.

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 (NC) or polyvinylidene difluoride (PVDF). The transfer step involves assembling a transfer “sandwich” (Figure 1) in which the gel is placed next to the membrane. Both are placed inside a cassette with blotting paper and sponges on either side to ensure a secure fit within the cartridge. Transfer occurs after 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.

Tools and reagents needed for membrane transfer:

  • Transfer tank: a tank system to carry out two transfers from mini gels to membranes. The Aqua Transfer Cell from Azure includes everything you need for the transfer step, like two cassettes and cooling units. The cassettes and electrodes are also color-coded to make it easier to set up the transfer with the correct directionality.
  • Power Supply supplies power to the tank. The Azure Power Supply powers up to four different modules simultaneously and has a built-in feature that allows you to create and save protocols for electrophoresis and Western blotting.
  • Pre-cut Blotting Paper is used for transfer sandwiches to save time and reduce waste. Choose from three sizes: 9 cm x 7 cm, 10 cm x 15 cm, and 13 cm x 18 cm.
  • Choose from two types of pre-cut membranes:
    • PVDF Membranes: can be used, stripped and reprobed without a loss of sensitivity or increased background
    • Nitrocellulose Membranes: high protein-binding affinity, typically used for chemiluminescent detection
  • Azure Transfer Buffer is formulated for enhanced protein transfer and improved sensitivity.
Wet transfer setup for electrophoresis
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.

Step 3: Blocking

Before the target protein(s) can be detected on the Western blot, non-specific binding sites on the membrane must be blocked. This is done by incubating the membrane in a blocking buffer. For ease of use, premade blocking buffers are available for purchase on the market. More details are found below. Homemade blocking buffers contain proteins, such as dry milk or serum albumin, to block non-specific protein-binding sites.

What you need for membrane blocking:

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.

What you need for primary antibody incubation:

  • Incubation Trays have lids to prevent dust or other contaminants from contacting the blot. Check out these incubation trays, which are available in clear or black, in a variety of sizes, that are perfect for washes.
  • Blot Washing Buffer is compatible with all chemiluminescent and fluorescent Western blots
  • Fluorescent Blot Washing Buffer is the best choice for near-infrared fluorescent Western blots because it is optimized to produce high signal-to-noise ratios with all fluorescent blots.
Pouring liquid inside black Western blot incubation box during Western blotting
Use opaque containers to protect the light-sensitive fluorophores during incubation. These opaque, black blot boxes are great for light-sensitive applications, like near-infrared fluorescent and chemiluminescent Western blots or protein gels.

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). HRP can react with a substrate to produce light (chemiluminescence), or a colored product that can be detected visually using an Azure Imager, another digital imager, or using film.

What you need for secondary antibody incubation:

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 a digital imager like the Azure 300 Imager, or using film in a dark room.

For fluorescent detection, the blot is imaged using an imager such as the Azure 500 that has a light source to excite the fluorophore and the correct filters to detect the emitted fluorescence.

What you need

  • Chemiluminescent HRP substrateL
    • 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
  • Using a chemiluminescent annotation pen, such as the ChemiWriter, enables you to write or draw on your transfer membranes, which results in a chemiluminescent signal on your markings once substrate is added.
  • Blot Development Folders: transparent plastic sheets to hold chemiluminescent blots during imaging, a wrinkle-free alternative to plastic wrap.

Step 6: Western 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 for Western blot analysis:

  • 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.
  • An analysis software like AzureSpot Pro software facilitates smooth Western blot analysis, while giving you the power to do background subtraction, band detection, and normalization.
AzureSpot Pro analysis Software
The background subtraction feature allows for more accurate analysis in certain situations, such as total protein normalization.

Need help with your Western blots and don’t know where to begin?

Let us help!

Scientist using pliers to place gel inside Azure chemiSOLO chemiluminescent imager
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Western Blotting Protocols

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