Western Blotting

The western blot (or immunoblot) technique has been a fundamental in protein analysis since the 1970s, the decade when it was first discovered that biomolecules could be spotted directly onto membranes (spot ELISA or DNA dot blots), or transferred from gels (southern blots, northern blots, western blots). Despite the now routine application of western blotting, successful results can depend on several factors including optimal membrane selection for the desired detection method and protocol. 

Western blotting is an analytical technique in molecular biology often used to investigate and characterize a protein’s post-translational modifications, for protein identification, and in protein production validation. Simple, yet effective, the western blot has applications in many settings including basic science research, biopharmaceutical production, forensics, and diagnostics.

The key to reliable and reproducible western blot results is a clean, low-background image. Problems can arise from:

• low tensile strength membranes 
• high background signal
• high membrane burn-through
• limited compatibility with detection methods 

While the steps in western blotting protocols remain the same, detection methods vary.

• The first step in a western blot is the separation of proteins via gel electrophoresis from a sample containing a mixture of proteins. Pall’s Nanosep^® centrifugal devices with low protein-binding Omega™ membranes are ideal to concentrate samples for gel electrophoresis. 
• Once separated on the gel, the proteins are transferred to a polyvinylidene fluoride (PVDF) membrane or a nitrocellulose membrane. This step immobilizes the proteins for further analysis. 
• The membrane is then incubated with labelled antibodies specific to the protein of interest. 
• After incubation, the membrane is washed to remove any unbound antibodies and further analysed by various immunodetection techniques.

The most commonly used detection methods involve radiolabels, fluorophores, chromogenic and chemiluminescent enzymatic reactions. Each approach has its own benefits and considerations: 

• Radiolabelled probes support protein detection directly via X-ray film; enhanced safety requirements are needed due to radioactivity.
• Chromogenic reactions do not need specialized detection equipment, color-formation occurs on the membrane and can be visualized with the naked eye. 
• Chemiluminescent reactions utilize light-sensitive equipment or materials to process and further analyze western blot results. 
• Fluorescent probes used as a detection method allow for multiplexing and concurrent detection of multiple proteins with different molecular weight.

We offer a variety of transfer membranes that have been optimized for western blotting applications. The sensitive nature of our membranes yields high resolution results with low background and burn through.

• Pall’s FluoroTrans® PVDF transfer membrane has been optimized for fluorescent detection to ensure a very low background that will not interfere with protein detection and analysis when exposed to fluorescence. Autofluorescence from standard western transfer membranes can obscure specific signals, especially at lower wavelengths, when using a fluorescent detection method.
• Pall’s FluoroTrans W PVDF  transfer membrane is optimal for use with traditional staining and chemiluminescent detection methods as it exhibits high sensitivity, low burn-through, and low background. 
• Pall’s BioTrace™ NT  transfer membrane is a pure nitrocellulose unsupported media that is compatible with a variety of detection systems andhas a high binding capacity for nucleic acids and proteins. This high protein binding capacity is ensured due to the homogenous nature of the membrane. Other nitrocellulose membranes contain high levels of cellulose acetate which reduce the protein binding capacity of the membrane.  Physical characteristics of BioTrace NT membrane such as high tensile strength and hydrophilicity provide excellent handling ensuring that the membrane will not rip, tear or crack during transfers.