Power wave 200 microplate scanning spectrophotometer

Ileum tissues were homogenized by mechanical disruption in RIPA buffer with a cocktail of protease inhibitors (P8340, Sigma, USA) and incubated on ice for 30 min. Lysates were centrifuged at 12000 rpm for 10 min and the protein content was stored at -20°C for further use. For detection of MLCK, MLCP and PKC, ninety-six-well microplates were incubated with antibodies against MLCP, PKC and MLCK at 4°C overnight. After four washes with PBS, the wells are saturated with BSA (200 μl, 3% in PBS, at least 2 h at 37°C). After four washes with PBS, 100 μl of sample containing MLCP, PKC or MLCK protein was deposited in the wells, and protein was allowed to bind to its specific antibody during 3 h at 37°C. After four washes with PBS, streptavidin-HRP (Beckman, USA) was added (100 μl, 1/1000 in PBS, 1% BSA, 30 min at 37°C). After four washes with PBS, 100 μl of HRP substrate are deposited and reaction was allowed during 10 min in dark (upon oxidation, TMB forms a water-soluble blue reaction product that can be measured spectrophotometrically at 650 nm). Reaction was then stopped by addition of 100 μl H₂SO₄ 2 M (upon acidification, the reaction product becomes yellow with an absorbance peak at 450 nm). Absorbance at 450 nm (yellow) was read by BioTek PowerWave 200 Microplate Scanning Spectrophotometer (Winooski, VT, USA).

96-well plates (MaxiSorp^® flat-bottom 96-well plate, Fisher Scientific Inc., Fair Lawn, NJ) were coated with 500 ng actin in 100 μl carbonate-bicarbonate buffer (pH 9.5) for 1 h at 37°C. The plate was washed once with TBS and then blocked with 3% BSA in 200 μl TBS buffer at 37°C, for 1 h. Wells were then washed once with 250 μl TBS. trT2-50 was added at 1:2 dilutions in 100 μl TBS, starting from 500 ng/well, incubated for 1 h at 37°C and then washed three times with TBS containing 0.1% Tween-20 (TBST). Each well was then treated with 100 μl rabbit anti-trT2-50 diluted 1:500 in TBS and incubated for 1 h at 37°C. The wells were washed three times with TBST and then incubated with 100 μl goat anti-rabbit IgG-HRP (Jackson ImmunoResearch, West Grove, PA) diluted 1:10,000 in TBS, for 1 h at 37°C. Wells were then washed twice with TBST, and once with TBS before 100 μl substrate (1-step Ultra TMB-ELISA, Pierce, Fisher Scientific Inc.) were added. Absorbance at 655 nm was measured 10 min thereafter using a Power Wave 200 Microplate Scanning Spectrophotometer (Bio-Tek Instrument, Winooski, VT). Affinity was evaluated by double reciprocal plot [28 , 29 (link)].