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Western Blotting

Optimize western blot transfer protocols for sharper images

Western Blot Protein Analysis with Clear Results

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.

 

 

Achieve Reliable and Reproducible Western Blot Results

 

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 
 
High quality membranes are imperative to assess the relative abundance of proteins for analytical purposes. Pall’s blotting membranes offer high tensile properties with low background levels. Our transfer membrane range supports a wide selection of detection methods without compromising quality.
 

What is Your Western Blot Detection Method of Choice?

 

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.
 
Both qualitative and quantitative results rely on the difference between the background and the signal from the protein of interest.
 

Transfer Membranes Optimized for Low Noise and Clear Results

 

 

laboratory-wester-blotting

 

 

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. 
 
Since the membrane is detergent free and unsupported, it yields sharper images with little to no distortion and has lower protein burn through than competitive nitrocellulose media in electrophoretic transfers. Highly consistent, both inter- and intra- lot, this membrane provides sensitive detection of biomolecules blot after blot.
 

Table: Western Blot Transfer Membrane Selection Guide

 

Material   

Nitrocellulose   

PVDF

Product

BioTrace NT

FluoroTrans PVDF

FluoroTrans W PVDF
Application

Colony/Plaque Lifts

 

Western Transfers

 

Protein Dot/Slot Blots

(Primary) N-terminal Protein Sequencing 

 

(Also) Western Transfers Protein Dot/Slot Blots

(Primary)Western Transfers

 

(Also) N-terminal Protein Sequencing 

Detection Method

Radiolabeled

Probes

 

Direct Stain,

 

Fluorescence

 

Enzyme-antibody

Conjugates

 

Chemiluminescent

 

Chromogenic

Fluorescence 

 

Radiolabeled

Probes

 

Direct Stain 

 

Enzyme-antibody 

Conjugates 

 

Chemiluminescent 

 

Chromogenic

Radiolabeled

Probes

 

Direct Stain 

 

Enzyme-antibody 

Conjugates 

 

Chemiluminescent 

 

Chromogenic

Features

No support fabric

 

No detergents added

 

100% pure nitrocellulose

Chemical resistance

 

Non-flammable

 

High strength

 

Strong protein binding

 

Very low burn-through

 

Sensitive detection

 

Good chemical compatibility

Chemical resistance

 

Non-flammable

 

High strength

 

Strong protein binding

 

Very low burn-through

 

Sensitive detection

 

Good chemical compatibility

Benefits

Easily wetted with aqueous solutions, for less reagent usage

 

Excellent strength

Recommended for N-terminal protein sequencing: coating-free

 

Low-fluorescence background

 

suitable for fluorophore detection

 

Highest protein affinity/avidity for optimal transfers

Ideal for most western blotting applications – except fluorophore detection

 

High sensitivity; low background

FluoroTrans® PVDF and FluoroTrans W PVDF Transfer Membranes

Optimize western blot transfer protocols for sharper images
Optimize western blot transfer protocols for sharper images
Read more

BioTrace™ NT Nitrocellulose Transfer Membrane

Optimize western blot transfer protocols for sharper images
Optimize western blot transfer protocols for sharper images
Read more