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Mechanisms of Tape Stripping and Protein Quantification
By: Ali Alikhan, MD, and Howard I. Maibach, MD
Posted: February 26, 2010, from the March 2010 issue of Cosmetics & Toiletries.
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Measuring Protein Samples
After the harvesting of SC onto adhesives is complete, several methods can be used to measure the collected protein. For decades, weighing (gravimetry) was the preferred method despite the inconvenience of weighing before and after stripping under constant hydration conditions. Additionally, results were subject to inflation secondary to absorption of exogenous (topically applied) or endogenous (sebum, sweat and interstitial fluid) substances within the SC; typically initial strips were most affected by this absorption.
Little more than a decade ago, a novel colorimetric method was developed and validated by Dreher et al.15 This method relies on a protein assay similar to one developed by Lowry et al. more than half a century ago. Lowry’s method involved the measurement of protein with a folin phenol reagent after alkaline copper treatment.16 It was demonstrated to be simple, sensitive, specific and easily adaptable to small scale analyses, making it suitable for the measurement of miniscule absolute protein amounts.16Dreher’s method relies on spectrophotometry and colorimetry based on the calibration of stained SC proteins to the corneocyte mass.17 Drawbacks of Dreher’s method include the time-consuming preparation of tape strips and the necessary destruction of the original strips.
The Bradford dye reaction, which relies on Coomassie Brilliant Blue G-250 dye, is similar to Dreher’s method. The dye binds protein, resulting in ionic and hydrophobic reactions with a spectral shift from reddish-brown to blue. The maximal absorption for the bound form of the dye is 595 nm, the optimal wavelength for colorimetric measurement once the reaction has occurred. Despite disadvantages (e.g., serial dilutions), it is a fast and generally reliable method for protein quantification.
Dreher’s colorimetric method has been successfully adapted to 96-well microplates, effectively shortening analysis time.18 It should be noted that limited areas of adhesive tape are not predictive of SC removal on the entire tape; therefore, SC distribution on the tape is not homogeneous.18
A pivotal study examined direct spectroscopic SC protein quantification via absorption in the visible range (595 nm and 600 nm), with and without staining of corneocyte aggregates, and in the UV range (278 nm).19 Correlation coefficients R2 were 0.71 and 0.74, respectively. The results demonstrated weak SC protein absorption with immense light scattering.19 The Coomassie Brilliant Blue protein coloring did not increase light absorption by SC proteins, and thus could not decrease interference secondary to light scattering.19 The absorption techniques utilized in this study could not accurately predict corneocyte aggregate quantity.