PART 1 OF A 3-PART SERIES ON RELIABLY QUANTIFYING WESTERN BLOTS
Can Western blots be trusted? This question has sparked much debate in the research world after being the focus of a recent column in Lab Times.1 The article addressed the growing number of published papers that have been retracted due to doctored results in experiments involving Western blots, and called for new standards for those who use the technique.
The article may have meant to simply raise a red flag, but it opened a floodgate of misgivings about the technique, leading to animated discussions in the lab and on the Internet.
“I’d like to see Western blotting follow its Northern cousin into oblivion,” commented one reader of the blog “Retraction Watch.”2 Another reader of the blog dissented, saying the onus should really be on scientists to conduct experiments with higher integrity: “a bad workman blames his tools.” And someone else chimed in with a similar message: “It’s up to the scientist to make the controls convincing and the result clear and reproducible.”
So where do we go from here? While Western blots are widely used in science, a recent survey conducted by Bio-Rad revealed that more than 40 percent of scientists who responded say their Western blots fail a quarter of the time. [See Infographic: Western Blots Can Be Reliably Quantified?3]
What will it take to make every researcher feel more confident about their Western blots?
Let’s take a closer look at where doubts arise with Western blots. One major concern is that experimentalists often don’t know if they’ve performed the technique successfully until they reach the final detection step.
Trust, but Verify
U.S. president Ronald Reagan adopted the Russian maxim “trust, but verify” when describing his dealings with the then-Soviet Union. Scientists should adopt this tactic when it comes to Western blotting. You can apply “trust, but verify” to two different steps: protein separation and protein transfer. When identifying the top reasons why blots fail, scientists who responded to Bio-Rad’s survey cited those steps. Yet 42 percent said they don’t take the precaution of confirming protein separation and loading before transfer to the membrane. And an even greater share of respondents—47 percent—don’t determine whether their proteins transferred to the membrane before probing with an antibody. For these researchers, trust trumps verification.
For those who don’t check the transfer step, how do they determine success? According to the western blotting survey, two-thirds relied on prestained standards. But prestained markers lack the complexity of typical experimental samples and tend to transfer with much higher efficiency. They also don’t uncover potential problems such as uneven transfer or bubbles. While using a prestained marker may offer convenience, scientists should not rely on it as a verification tool.
Of those who do “trust, but verify” the transfer step, more than half (52 percent) said they use reversible staining. But there are concerns here, too. While reversible staining is good practice, it also adds extra steps, time, and cost. Staining using a commercial kit can add well over an hour to the procedure. Of course, if you were diligent enough to check this step, you probably wouldn’t have neglected to check the protein separation step. When you combine the time it takes to check both separation and transfer steps, you’ve now added a total of three hours to a protocol that altogether typically averages 18 hours. You’re a hard-working scientist, not a 7-Eleven. The immense time commitment notwithstanding, some also worry that reversible staining interferes with their western blot results.
A Better Way to Reliable Results
What’s a researcher to do? On one hand, you don’t want to skip the steps that can help you verify results and ensure that your experiment is proceeding according to plan. But as we’ve established, staining is a hassle.
There is an alternative that makes it easy for every researcher to verify each of these steps in less than three minutes. Bio-Rad has introduced a compound that binds to proteins during electrophoresis and fluoresces when activated by a “stain-free” enabled imaging system4, allowing for gels to be quickly examined after electrophoresis and membranes after transfer.
These checkpoints are part of a complete system of tools called the V3 Western Workflow5 that dramatically reduces the time to complete each non-antibody step in the workflow, including protein separation and transfer. (See Infographic: Traditional and V3 Western Workflow6)
By adopting a “trust, but verify” mentality using stain-free technology, every scientist can be more confident in their Western blotting process.
In part two, we explore the often-overlooked drawbacks of using housekeeping proteins. Part three looks at the common misperceptions of using film. For each “problem,” we’ll present solutions and alternate approaches to reaching your research goal. By the time you’ve completed all three segments of this special series, you’ll have the knowledge to approach your western blots and their quantification with confidence. Trust, but verify, us.
For more information on western blotting methods, ideal transfer conditions, and troubleshooting western blots, visit Western Blotting - Applications and Technologies.
1. Marcus A, Oransky, I. Can we trust western blots? Lab Times. 2012; 51. http://www.labtimes.org/labtimes/issues/lt2012/lt02/lt_2012_02_41_41.pdf. Published February 2012.
2. Oransky I. Can we trust western blots? Retraction Watch. http://retractionwatch.wordpress.com/2012/04/03/can-we-trust-western-blots. Published April 3, 2012.
4. Gilda JE, Gomes AV. Stain-Free total protein staining is a superior loading control to β-actin for Western blots. Anal. Biochem. 2013; 440 (2) doi: http://dx.doi.org/10.1016/j.ab.2013.05.027. Published September 15, 2013.