Calibration of 2‐D soil zymography for correct analysis of enzyme distribution

Abstract

Soil zymography is a new technique developed to visualize two‐dimensional distributions of enzyme activities. The method consists of incubating a membrane saturated with an enzyme‐specific fluorogenic substrate on a surface of the soil sample, followed by recording the membrane image generated by a fluorescent product (e.g. MUF: methylumbelliferone) in ultraviolet light. Despite its relative ease of use, performing zymography involves multiple user‐made decisions that might affect the accuracy of enzyme activity estimates. Therefore, unification of the zymography methodology is required for correct estimations and comparisons of various studies. We evaluated the following methodological aspects of the implementation of zymography: (a) camera settings and image processing, (b) effects of evaporation and (c) calibration procedures. Camera settings (shutter speeds or exposure time) affected the intensity of background fluorescence and signal‐to‐noise ratios (SNR). However, because their combined effects varied depending on MUF concentrations, light and camera setting need to be optimized for the expected range of MUF concentrations prior to zymography. Evaporation of MUF solution from the membrane had no effect on fluorescence. Relations between MUF concentration and intensity of fluorescence during calibrations demonstrated a saturated pattern and were strongly affected by image noise outside the optimal range (e.g. 8–14 μm MUF pixel−1). We developed a new calibration approach that is based on a piecewise linear regression. The new approach accounted for specific ranges of MUF concentration and uses nonuniformly saturated membranes, reflecting the real distribution of enzyme activities in soil. The new calibration algorithm eliminated biases of the standard calibration and resulted in greater accuracy in predicting MUF concentrations. Highlights We developed a new approach to calibration for 2‐D soil zymography. The approach accounted for spatial nonuniformity of soil zymograms. Standard calibration resulted in systematic underestimation of enzyme activity. Soil zymography requires pixel‐based calibration with nonuniformly saturated membranes.

A Visual Leaf Zymography Technique for the In Situ Examination of Plant Enzyme Activity under the Stress of Environmental Pollution

“…The membranes were placed on a black flat field in a lightproof room and illuminated by UV radiation with an excitation wavelength of 355 nm and emission wavelength of 460 nm . Camera settings (Fujifilm X-T20, XF 18-55 mm, f/2.8, 1/125 s, ISO-12800) and the distance between the camera and the samples (40 cm) were fixed to ensure constant photographic conditions …”

“…The fluorescent tag of each substrate (MUF) was dissolved in 0.1 M MES buffer to create a series of calibration concentrations (i.e., 0, 4, 8, 10, 15, 20, 40, 80, 100, 200, and 400 μM). Then, 15 μL of the MUF was applied to the center of the membrane using a micropipette . The membranes were imaged under the same UV radiation and photographic conditions.…”

“…10 Camera settings (Fujifilm X-T20, XF 18-55 mm, f/2.8, 1/125 s, ISO-12800) and the distance between the camera and the samples (40 cm) were fixed to ensure constant photographic conditions. 32 To quantify the enzyme activities of zymograms, a standard calibration was prepared to calculate the enzyme activities of different fluorescence intensities related to the gray values of the pixels of a zymogram. 33 The calibration was performed using the prepared 2 × 2 cm membranes that were soaked in an MES solution for 10 min and air dried for 2 min.…”

“…Then, 15 μL of the MUF was applied to the center of the membrane using a micropipette. 32 The membranes were imaged under the same UV radiation and photographic conditions.…”

Editorial for soil organic matter 2019 special issue

“…The first four papers focus on fundamental and methodological aspects of SOM. Guber, Kravchenko, Razavi, Blagodatskaya, and Kuzyakov () present a methodological paper on the new technique of soil zymography, developed to visualize two‐dimensional distributions of enzyme activities using fluorescence. This novel technique requires significant decision making by the user, and the authors discuss various options to exploit the exciting potential for capturing the spatial and temporal dynamics of enzymes in soil.…”