With the increased interest in proteomics and the study of protein interactions, sample integrity has become more important to ensuring accurate analysis. Protease enzymes can cause damage to protein samples, destroying experimental data that may have been time consuming to collect. The Sigma Protease Fluorescent Detection Kit is a tool that enables the detection of protease contamination via a fluorescent signal. The assay works when Fluorescein Isothipcyanate (FITC) labeled casein is cleaved into fragments by proteases. Upon acidification of the incubated samples with tricholoroacetic acid (TCA), digested fragments remain in solution while the uncleaved FITC-casein substrate is separated out by centrifugation. A fluorescent signal from the supernatant is an indicator of protease contamination.
The kit includes all of the necessary components for detection including incubation buffer (for the primary part of the assay), assay buffer (for detection), FITC-casein substrate, TCA solution (for sample acidification), a FITC fluorescent control and trypsin for a positive assay control. To prevent damage from freezing and thawing, the FITC-substrate should be stored in smaller aliquots than the 5ml provided. The trypsin control requires resuspension prior to use and may be stored frozen for only 4 weeks. All other kit components are ready to use.
The assay procedure itself is fairly straightforward and will take approximately 3-4h, depending on the skill level of the user and the number of samples to be analyzed. To begin, the samples are mixed with incubation buffer and the FITC-casein substrate. After a 60 min incubation at 37oC, the samples are acidified with 0.6N TCA and incubated for another 30 min at 37oC. Samples are then centrifuged for 10 min at 10,000 x g, mixed with the assay buffer and fluorescence is measured in the supernatant at 485 nm excitation and 535 nm emission. I found the best method for separating the samples from the precipitate to be centrifugation at 12,500 x g, in spite of the suggested 10,000 x g speed. If not completely separated, the uncleaved FITC-casein substrate will contribute to the final fluorescent measurement of the supernatant, yielding a false positive. The kit is somewhat flexible allowing samples to be analyzed in either a cuvette or a multiwell plate.
Overall the assay is fairly simple, can be used for a braod range of sample types and does not require significant training (with the exception of reagent handling). The user must take care as the FITC-casein is sensitive to light and temperature. In addition, contamination of other reagents or samples by trypsin must be prevented. Reagents with dark dyes (would block the fluorescein or protease inhibitors), solutions using high concentrations of detergents or solutions with pH extremes are incompatible with this kit. When using dilution series standards of the trypsin control, the results are quantitative with a moderate deviation. If the assay is performed carefully and the centrifugation process is optimized, quality results are obtainable. I did not find this assay to be as accurate or easy to use as other simple fluorescent/quencher cleavage systems however, it is sufficient for diagnosing general protease contamination levels.
The kit quantities enable the evaluation of 250 samples (limited by the 5ml quantity of FITC-casein substate). The price of the kit is $185.00, broken down that is less than $1.00 per sample, thus the kit is very reasonably priced. The benefits of this kit include the versatility of sample types that may be used and cost effectiveness. The drawbacks include the moderate level of complexity and handling requirements, although quality control can ensure good results. Overall, I would recommend this kit as a way to detect general levels of protease contamination in reagents or samples however, I would not recommend this kit for QC or final product analysis.
Daniel J. Schwartz
Scientist
Gentel Biosurfaces, Inc.
Madison, WI