Are you using Western blots for quantitative comparisons of protein expression? If you are, you will need a way to reliably evaluate your experimental outcomes. Comparing protein abundance means taking the definition of quantitative a bit further.
Considerations to Compare Western Blot Data Accurately
To compare relative protein abundances accurately, you first need to:
- Ensure signal and protein expression are proportional by detecting within the linear range.
- Choose the right normalization strategy for your assay to account for intrinsic variability.
- Perform each assay under the same experimental conditions.
Then you will use the quantitative data you generated with each blot and compare them. Proper analysis takes all factors into consideration and reduces variability wherever possible. The process can be challenging but is absolutely necessary to get published. Find additional resources to improve your quantitative Western blot results.
“Most importantly, it is possible to compare two blots only if they present exactly the same conditions, using different lysates derived from cells cultured and treated in the same way.”
Degasperi A, Birtwistle MR, Volinsky N, Rauch J, Kolch W, Kholodenko BN. (2014). PLoS ONE, 9(1). DOI: 10.1371/journal.pone.0087293
Multiple Film Exposures
Multiple film exposures from the same blot will inevitably be captured under differing conditions. This could be due to exposure time, enzyme/substrate kinetics, or many other factors. This means the data you depend on to make accurate comparisons, are constantly changing. What’s more, film detects signals with a lack of proportionality in both high and low intensity signals. This is a result of the Reciprocity Law. Faint signals end up being underrepresented, while strong signals can overpower film. For these reasons, it is not advised to compare multiple film exposures or to combine data captured on different pieces of film. Similar effects are observed when chemiluminescent Western blots are imaged on traditional CCD-camera based instruments, although to a lesser extent.
Stripping and Reprobing
Stripping is not uniform across the entire blot. This creates additional variability which hampers your ability to make accurate comparisons. Light stripping creates artifacts and harsh stripping leads to a loss of protein sample. For these reasons, stripping and reprobing is not recommended for reliable quantitative Westerns.
Multiplexing
If you’re looking for the most accurate way to compare protein abundance, a near-infrared imaging system with the ability to multiplex, such as the Odyssey® CLx Imaging System, is the way to go. Multiplexing is dual color detection that allows you to simultaneously detect two targets in one sample on the same blot. This is done by using distinct fluorescent dye-labeled secondary antibodies. By combining near-infrared detection with multiplexing in an imaging system with a wide dynamic range, you get high sensitivity with no saturation and improved consistency. This allows you to detect both faint and strong signals and account for lane-to-lane variation in loading and transfer without the need to strip and reprobe. You are able to get the full story from a single acquisition. Find out more about multiplexing and the Odyssey CLx Imager.
“A major advantage of using fluorescently labeled secondary antibodies is the ability to detect multiple targets by using fluorophores with nonoverlapping excitation-emission spectra.”
Ghosh R, Gilda JE, Gomes AV. (2014) Expert Review of Proteomics. 11(5):549-560. DOI: 10.1586/14789450.2014.939635
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