Editorial Article
Monday June 22, 2009
by Caitlin Smith
Though there are many ways to detect proteins, the decades-old technique of western blotting remains a protein detection mainstay. “Western blotting is the best way to detect one protein within a mixture of other proteins, if you know what you are looking for,” says Lisa Isailovic, marketing manager at Alpha Innotech. But this mainstay continues to evolve to meet scientists’ demands. Researchers are now seeing strides in the quantification, sensitivity, and time savings available in today’s technology for western blotting.
Quantification with fluorescent westerns
While chemiluminescence is better for detecting rare signals in westerns (see below), fluorescent westerns enable the relative quantification of different signals, using secondary antibodies coupled to fluorophores. “Compared to older techniques, such as chemiluminescence or colorimetric detection, fluorescence signal is easier to generate (there are no substrates) and the signal is immediate and stable,” says Megan Schmidt, product manager for imaging agents at Carestream Molecular Imaging. “Fluorescence detection is not an enzymatically driven reaction and is more quantitative. Also, with fluorescence, you can utilize different colors to image multiple proteins on the same blot.”
Carestream Molecular Imaging recently released their X-SIGHT Western Kits, which use X-SIGHT Nanosphere Conjugates. These are antibody-conjugated nanoparticles loaded with fluorescent dye. Schmidt says that this reagent “delivers a more intense signal, with better photostability, than other fluorophores. That means you are able to detect proteins of lower abundance and image a blot more times than with other common fluorophores.” Schmidt is also excited about X-Sight Nanospheres that emit signals in the IR and near-IR range. “Background was improved with the use of dyes that emit in the near-IR to IR range, avoiding autofluorescence of membranes and some cellular proteins,” she says. “Encapsulating these dyes inside a nanoparticle increases the brightness and stability of these IR fluorescent compounds.”
Fluorescent western blots can also help you normalize your data. “Fluorescent westerns enable you to image overlapping proteins on the same blot, without stripping and re-probing,” says Isailovic. “They also let you use one or two colors for detecting your proteins of interest, and a third channel to detect a protein like actin that will let you normalize your protein of interest to a loading control.” Alpha Innotech’s new SpectraPlex Fluorescent Western Kit was designed to help new fluorescent western blot users convert from chemiluminescent to fluorescent westerns. Their new kit “contains the reagents needed to easily perform two color westerns blots, and when combined with the FluorChem Q, is able to detect femtograms of protein,” explains Isailovic. FluorChem Q is their western blot imaging system, also designed for this conversion to fluorescence. “On one imaging system, you can use chemiluminescence to detect low-abundant proteins, and fluorescent westerns for quantitative multi-protein detection,” says Isailovic.
Priya Rangaraj, product manager at Thermo Scientific Pierce, agrees that “one of the most exciting new developments lies in multiplexed, fluorescent western blotting techniques. New Thermo Scientific DyLight Dyes are now available that offer sensitivity comparable to chemiluminescence.” Thermo’s two DyLight™ Fluorescent Western Blotting Kits include different pairs of these dyes. “DyLight 680 and 800 dyes that facilitate detection in the infrared range are particularly attractive, as they limit the autofluorescence from biological samples,” says Rangaraj.
Sensitivity
Despite the advantages of fluorescent westerns, chemiluminescent westerns are not likely to become obsolete soon. “Chemiluminescent western blotting still has a long life ahead of it as the most sensitive detection methods for confirming the presence of small amounts of protein,” says Isailovic.
Isailovic notes that the use of CCD imaging systems for western blot detection is becoming more attractive to those needing greater sensitivity. “Only within the last few years has CCD technology advanced enough to equal film for both speed and sensitivity,” says Isailovic. “However, scientists have been slow to uptake this change because of the perceived notion that using an imaging system will not give them the sensitivity required for their research. This perception is beginning to change, however, as more scientists are exposed to imaging systems like the FluorChem HD2 or FluorChem Q, and use them on a day-to-day basis.” Craig Smith, product manager for in vitro instrumentation at Carestream Molecular Imaging, also notes that when “using digital imaging vs. film imaging, the image saturations present on film can be controlled for using saturation settings that meter the light release of the sample and stop image collection when saturated.”
LI-COR Biosciences combines its infrared expertise with chemiluminescent westerns in their Chemi-IR™ Detection Kit, which “utilizes an anti-HRP tertiary antibody conjugated with the IRDye800CW near-IR (NIR) fluorophore,” says Tony Endozo, marketing product manager at LI-COR Biosciences. “This product bridges the technologies between the indirect detection method of chemiluminescence and the direct detection methods of NIR offered by LI-COR.” Western blots can be subsequently imaged on LI-COR’s Odyssey® Infrared Imaging System. The Chemi-IR™ Detection Kit offers a method to obtain direct quantitative data with chemiluminescent detection, and may also be helpful for researchers using chemiluminescent westerns who might encounter difficulties optimizing the near-IR blots. “Oftentimes the reason for an ineffective switch from chemiluminescence to near-IR has been finding that right secondary for, especially, those esoteric primary antibodies,” says Endozo.
Indeed, one of the biggest challenges in western blotting, according to Rangaraj, is finding reliable primary antibodies. “There is a strong need for antibodies validated specifically for western blotting techniques,” says Rangaraj. “To that end, [Thermo has] introduced more than 100 siRNA-validated antibodies for western blotting and, with our recent acquisition of ABR-Affinity BioReagents, we have greatly expanded our antibody offerings.”
Beating the clock
Why wait longer than you need to for a western? Millipore says you don’t have to, with their SNAP i.d. Protein Detection System, which speeds the protocol. “Millipore has developed tools such as the SNAP i.d. Protein Detection System to change the role western blotting plays in protein research,” says Sami Barghshoon, product manager for protein detection at Millipore. “What used to take four hours can now be done in 22 minutes.” Barghshoon says that it is not so easy for people to wrap their minds around this. “One challenge is to overcome the scientist’s preconceptions of the role of immunodetection and western blotting,” says Barghshoon. “Then there’s another challenge of getting scientists to digest that, and to adopt the new paradigm. For instance, the SNAP i.d. system is not just about speed. It represents a paradigm shift—one that can significantly shorten research time, bringing discoveries closer than previously thought.” The SNAP i.d. system achieves its speed using a vacuum to drive reagents through the blotting membrane—this differs from traditional western blotting, in which reagents move by diffusion.
The Thermo Scientific Pierce Fast Western Blot Kit with ECL Substrate also reduces western blotting time significantly. “Using the new reagent-based Pierce Fast Western Blot Kit, scientists can perform a typical western blot in 55 minutes,” says Rangaraj. “The kit provides all the necessary reagents to complete a western blot that was probed with a mouse or rabbit primary antibody, yielding results comparable to classical methods. We also plan to introduce a SuperSignal Fast Western Kit in the near future.”
Whether you need faster blotting, greater sensitivity, or quantification, your data are likely to benefit from recent advances in western blotting.