Fluorescent Western Blotting Detection

Fluorescent reagents are growing in popularity for Western Blotting because they offer increased time savings over chemiluminescence Western Blot detection and reduced chemical waste compared to both chemiluminescent or chromogenic detection systems.

Fluorescent Western Blot applications differ from other detection systems in that the signal is not a product of an enzyme reaction, but rather a transient light emission resulting from the excitation and subsequent release of photons as the excited molecule returns back to its normal state. This is in contrast to enzyme-substrate systems that produce a colored product or light emission as a result of an chemical reaction.

Fluorescent Western Blot detection
Fluorescent Western Blot detection and results

Advantages of fluorescent Western Blot detection:

• Ability to multiplex Use of multiple fluorophores for simultaneous detection of several target proteins makes stripping and reprobing unnecessary.
• Increased dynamic range A 10-fold increase in dynamic range over chemiluminescent detection offers better linearity within detection limits.
• More quantitative Fluorescent detection is more quantitative than enzyme-based detection methods.
• Excellent stability Most fluorescent molecules provide excellent stability, allowing blots to be archived and re-imaged at a later date.

Imaging options for fluorescent Western Blot detection

It requires some appropriate imaging instruments in order to record and document the results of Western Blotting successfully. For fluorescent Western Blot, X-ray film is not suitable; digital imaging solutions are used to to capture signal such as charge-coupled device (CCD) camera-based imagers and scanner-based systems.

Imaging options for fluorescent Western Blot detection

  CCD camera-based imager Laser scanner-based system X-ray film
Suitable Yes Yes Not
Strengths • Broad linear dynamic range
• High sensitivity
• Quantitative imaging
• Digital image produced
• Broad linear dynamic range
• High sensitivity
• Quantitative imaging
• High resolution

Using loading control antibodies in fluorescent Western Blot detection

Owing to fluorescent Western Blotting, it's possible to quantitatively analyze protein of interest. Usually, loading controls which exhibit high-level, constitutive expression in the cell type or sample you are examining are used to normalize the Western Blot results. It is important that the protein chosen as a loading control has a different molecular weight than the protein of interest so that the bands are distinct and expression levels quantifiable. Popular loading control detection antibodies include anti-β-Actin monoclonal or polyclonal antibodies, anti-COX-4, anti-GAPDH, anti-Tubulin and anti-VDAC/Porin antibodies.