Precipitation of proteins was performed to remove salts from the sample, which affect the iso-electric focusing step.
After rehydration the strips were moved to a 12-manifold and iso-electric focusing was performed on an IPGphor II (GE Healthcare, Uppsala, Sweden).
To investigate additional physicochemical properties of the enzymes of interest, we subjected the plaque supernatant protein mixture to liquid iso-electric focusing (IEF).
In addition, gliadin zymography was conducted to obtain the approximate molecular weights and pH activity profiles of the gliadin-degrading oral enzymes and liquid iso-electric focusing was performed to establish overall enzyme iso-electric points.
The lumen fraction was concentrated using Microsep 3K centrifugal devices (PALL Life Ccience) and the buffer was diluted with double distilled water approximately to half the strength in order to decrease the salt concentration prior to iso-electric focusing (IEF).
Gliadin zymography revealed that prominent enzymes exhibit molecular weights >70 kD and are active over a broad pH range from 3 to 10. Liquid iso-electric focusing indicated that most gliadin-degrading enzymes are acidic in nature with iso-electric points between 2.5 and 4.0.
The tissue was assayed by two methods: Iso-electric focusing (IEF) and ER-ICA an immunocytochemical assay.
Iso-electric focusing was then performed at 20 °C for a total of 85 000 Vh using the Ettan II IPGphor system (GE Healthcare).
