Streptavidin horseradish peroxidase hrp

Proteins were separated on SDS-PAGE (15%) and transferred to a Hybond-P membrane (Amersham Biosciences) at 100 V for 1 h in transfer buffer. The membrane was then blocked with BSA at 4 °C overnight. After incubation with anti-His (Amersham) followed by secondary antibody incubation, the membrane was treated with a mixture of supersignal (Thermo, Waltham, MA, USA) west-femto substrate and west-pico substrate (1 : 5 vol/vol). Pierce imaging film was used and developed with a Compact 2 processor. Activity probe labelling was carried out in 100 mM NaCl, 25 mM NaOAc, 3 mM DTT, pH 5.5, using biotin-DEVD–FMK at 2–50 μM final concentration, at 37 °C for 60 min. Protein were separated on SDS-PAGE (15%) and transferred to a Hybond-P membrane (Amersham Biosciences). After incubation with streptavidin-horseradish peroxidase (HRP) (Amersham), the biotinylated proteins were detected using a mixture of supersignal (Thermo) west-femto substrate and west-pico substrate (1 : 5 vol/vol). Apparent size was calculated using GelAnalyser (www.gelanalyser.com); kDa brackets are given when apparent sizes varied slightly between gel repeats, as expected.

Poloxamer 407 (BASF, the Netherlands), PBS (phosphate buffered saline, 10 mM phosphate, pH 7.4, 140 mM NaCl, Fresenius Kabi, the Netherlands), IMDM (Iscove's modified Dulbecco's medium, Lonza, Belgium), human elastase (EPC, Missouri, USA), cytochalasin B (Sigma, Germany), N-formyl-Met-Leu-Phe (fMLP; Sigma, Germany). PTG (PBS supplemented with 0.1% Tween 20 and 0.2% gelatin A), TMB (3,3′,5,5′ tetramethylbenzamidine, Uptima INTERCHIM, France), streptavidin horseradish peroxidase (HRP) (Amersham Life Science, UK), 96-well maxisorb plate (Nunc, Denmark), 96-well polysorb plate (Nunc, Denmark). Biacore buffer HBS-PE (GE Healthcare, the Netherlands). Ampex red (Molecular Probes/Thermo Fisher Scientific, US), HRP (Sigma, Germany).