Although modern approaches to western blotting follow the same principles pioneered by the inventors of the technique—namely the separation of proteins on a gel and subsequent transfer to a membrane, followed by antibody-based detection—researchers now have access to many new innovations. Designed to make western blotting faster, easier, more accurate, and increasingly data-rich, these include novel approaches to enhance western blot detection such as Stain-Free imaging technology, fluorescent western blotting, and state-of-the-art developments in digital imaging. This article explains how such advances support better identification and quantification of proteins using a method that has been around for decades and offers guidance to help you determine which detection method is right for you.
Total protein staining is an established method to confirm that proteins have transferred successfully from a gel to a membrane. However, commonly used anionic dyes have low sensitivity (Ponceau S) or can interfere with downstream immunostaining (Coomassie Blue), as well as extending already lengthy western blotting protocols by introducing time-consuming staining and de-staining steps. Designed to address these issues, Stain-Free imaging technology utilizes acrylamide gels that contain a proprietary trihalo compound able to covalently bind tryptophan residues in the sample upon brief UV activation. Used in conjunction with a Stain-Free enabled imaging system, Stain-Free methods provide rapid verification of protein transfer without compromising downstream results and prevent wasted time by identifying problems early in the western blot workflow.
Quantitative western blots are widely used to compare target protein expression between different samples and rely heavily on normalization to correct for any inconsistencies in sample preparation, pipetting, and protein transfer. Conventionally, researchers have used a housekeeping protein (HKP) such as β-actin, β-tubulin, or GAPDH as a loading control, with the assumption that the expression level remains constant, yet HKP expression may vary considerably under different experimental conditions. Moreover, because HKPs are typically expressed at much higher abundance than target proteins, loading sufficient sample to detect the target of interest can often result in over-loading the HKP, taking the intended normalization signal out of the linear range and consequently rendering it unusable. Stain-Free total protein measurement serves as a more reliable loading control than HKP expression, providing truly quantitative western blot data by normalizing the target protein signal to total protein in each lane without the need for the added steps of HKP antibody incubation.
Although the earliest western blots relied on radiolabeled Protein A for detection, this practice was rapidly superseded by the use of antibodies labeled with enzymes such as horseradish peroxidase or alkaline phosphatase. Pairing these reagents with specialized substrates to produce measurable electrochemiluminescence provided researchers a far safer alternative to using radiolabels. However, a drawback of enzyme-based detection is that it produces only a finite signal and it is often linked to poor experimental reproducibility due to the kinetic nature of the detection reaction.
Fluorescence-based western blot detection has become increasingly popular in recent years since it delivers a more accurate representation of target protein abundance through not being bound by “signal amplification” due to enzyme substrate kinetics. Fluorescent western blotting also allows for blots to be cataloged for extended periods of time when stored appropriately, and opens up opportunities for multiplexing—by combining different antibody species with fluorophores of varying wavelengths, researchers can now detect several targets in parallel for deeper biological insight.
Another early western blotting practice that is slowly being phased out is the use of photographic film for imaging. Despite providing extremely high sensitivity, film suffers from a limited dynamic range, an inability to deliver quantitative data, and high associated costs for film development and waste disposal. It also necessitates laborious annotation that introduces the risk of experimental data being misinterpreted. Modern digital imaging systems combine the sensitivity of film with a wide dynamic range (≥ 4 logs) and superior signal-to-noise, making them a valuable addition to any laboratory.
Along with intuitive and highly accurate data quantitation, better environmental credentials (no chemicals), and no requirement for costly consumables (film), digital imaging also affords electronic annotation and data storage for improved experimental reproducibility and traceability. With publisher guidelines for analysis and submission of data from immunoblotting experiments becoming stricter, a digital imaging system represents a sound investment, with many platforms typically recovering the initial capital layout in under a year.
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Bio-Rad supports all your western blotting requirements, including Stain-Free imaging, fluorescent western blotting, and digital image capture and analysis. More information can be found here.