A3574

Photobleaching was measured similarly as previously described^{73 (link)}. A final concentration of 1 µM of each variant was embedded in polyacrylamide gels (168 µl 30% acrylamide/bis-acrylamide, 25 µl PBS, 0.5 µl TEMED, and 3 µl 10% APS (Sigma Aldrich, #A3574, #110-18-9 and #SML2389, respectively) and 57 µl fluorescent protein in PBS) inside microscope slides (ibidi, µ-Slide 8-well). Slides were mounted to Eclipse TI-E Nikon inverted microscope (Nikon Instruments Inc., Melville, NY) with Plan Apo DIC 60X/1.4 NA objective and equipped with a cooled electron-multiplying charge-coupled device camera (IXON ULTRA 888; Andor). The measurement consisted of six repetitions of exposure to the strongest available LED light at either 405 or 488 nm for 15 min while capturing an image every five seconds. Images were analyzed using ImageJ to recover the mean intensity from each frame. A bi-exponential function was fitted to normalized brightness as a function of exposure time. The weighted average of the exponential coefficients was calculated. Outliers were removed, and at least three measurements were used to calculate means and standard deviations.

SDS-PAGE and Tricine-SDS-PAGE electrophoresis of milk, bacterial proteins, and hydrolysates after simulated digestion were performed according to the protocols of Laemmli (1970) and Schägger and von Jagow (1987) [29 (link),30 (link)]. Briefly, for SDS-PAGE 12.5%, polyacrylamide gel was used; for Tricine-SDS-PAGE, the separating gel contained acrylamide and bisacrylamide at a 16.5:3 ratio, and the stacking gel at a ratio of 4:3 was made of acrylamide/bis-acrylamide solution (A3574; Sigma). Separation was conducted in a Mini PROTEAN3 Cell apparatus (Bio-Rad, Warsaw, Poland). The buffer in the Tricine method differed with the addition of Tricine to the Tris–buffer (39468; Sigma). Separation was conducted at 30 V for 30 min and 100 V for 120 min. A molecular weight marker Odyssey^® MW ranging from 10–250 kDa (928–40,000, Li-COR Biotechnology, Bad Homburg, Germany) was used, and the process was followed by Coomassie Brilliant Blue (R-250; Serva, Heidelberg, Germany) staining and visualization in a ChemiDoc Imaging System (Bio-Rad, Warsaw, Poland) equipped with the Image Lab (Bio-Rad) software.