Protein analysis was performed by SDS PAGE and immunoblot. SeeBlue® Pre-Stained Standard or SeeBlue® Pre-Stained Standard Plus2 (INVITROGEN) were used as standards for protein size in SDS PAGE. The identities of the recombinant C. oncophora and O. ostertagi depsiphilin N-termini were also confirmed by MALDI-MS.
5.23.1 Conventional Methods 5.23.1.1 Conventional SDS PAGE
Resolving SDS gels of 8 % and 10 % were either precast and stored in a moist milieu at 4° C or freshly poured. The stacking gel was pou red immediately before use. The samples were mixed with 0.1 volume of SDS Loading dye and then boiled for 5 min.
The samples were loaded onto the gel, and the gel was run for 20 min at 70 – 80 V and for approx. 1 h at 120 – 130 V. When the blue dye was near the bottom of the gel, the run was stopped and the gel was blotted.
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5.23.1.2 Conventional Western Blot
The sandwich for a conventional blot of SDS gels consisted (from anode to cathode) of 2 – 3 pieces of filter paper, a nitrocellulose membrane, the gel, and another 2 – 3 pieces of filter paper. The membrane and the filters were soaked in Blotting buffer for Western Blots before building the sandwich. A current of 100 mA was applied for 1 h in a SemiDry Blotting Chamber.
5.23.2 NuPage® System 5.23.2.1 NuPage® Gels
INVITROGEN offers a number of precast gels which can be run at higher voltages and therefore in a shorter time than conventional SDS gels. In this work the NuPage® Novex Bis-Tris 4 – 12 % gels were used. The resolution of proteins is better than in a conventional SDS gel. 3 part of sample was mixed with 1 parts of LDS sample buffer and heated to 70° C for 10 min. The gels were run i n the XCell SureLockTM Mini-cell in 1 X MES or 1 X MOPS at 200 V for 35 – 60 min.
5.23.2.2 NuPage® Western Blot
The NuPage® blotting sandwich was assembled into the blotting chamber of the NuPage® XCell SureLockTM Mini-cell. The sandwich consisted (from anode to cathode) of 2 – 3 filter pads, a filter paper, a nitrocellulose membrane, the gel, a filter paper, and 2 – 3 filter pads. The filter pads and the membrane were soaked in NuPage® Blotting buffer, and the filter papers were wetted with NuPage® Blotting buffer immediately before building the sandwich. When the sandwich assembly was fixed in the chamber, the whole sandwich was covered with NuPage® Blotting buffer, and the outer chamber was filled with deionized water. The blot was run at 200 V for 1 – 2 h.
5.23.3 Coomassie Staining
After blotting the gel was usually stained in Coomassie Staining Solution to visualize the protein. The gel was incubated in approx. 30 ml Coomassie Staining Solution for approx. 1 h under gentle motion. The unbound Coomassie Blue was removed by
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several washes in Coomassie Stripping Solution. The gel was then washed once in deionized water.
5.23.4 Drying SDS Gels
When a Coomassie-stained gel was to be saved, it was dried. For this approach it was placed between two layers of wetted Gel Drying Film (PROMEGA). This sandwich was fixed in a frame. After two days the Gel Drying Films and the gel were dry and could be excised.
5.23.5 Immunoblot
Following the protein transfer the membrane was immunoblotted. During the entire immunoblot the membrane was rocked gently. First it was washed for 5 min in 1 X TBS. Blocking was performed by incubating the membrane for 1 h in 1 X Roti®-Block Blocking Reagent (ROTH). Then it was washed 3 – 5 X for 1 – 5 min with TBS Tween. The incubation with primary antibody was performed for 1 h, the concentration depending on the antibody (see 8.4.4.1). Another washing procedure of 3 – 5 X with TBS Tween for 1 – 5 min each followed. The membrane was incubated with the secondary antibody for 1 h and then washed as described above.
A final wash with 1 X TBS without Tween followed. The concentration of secondary antibodies is given in 8.4.4.2.
5.23.5.1 Alkaline Phosphatase
Most of the secondary antibodies used were conjugated with Alkaline Phosphatase (AP), so the blots were developed with an NBT / BCIP substrate (Staining Solution for Western Blots), which gives a violet to black color. The reaction was stopped by washing the membrane in deionized water.
5.23.5.2 Horse Radish Peroxidase
For some approaches a more sensitive detection system was necessary. The SuperSignal West® Femto Maximum Sensitivity Substrate Kit (PIERCE), based on chemiluminescence, detects secondary antibodies coupled with Horse Radish
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Peroxidase (HRP). After incubation with the secondary antibody the membrane was washed 3 – 5 X with TBS Tween and then 3 – 5 X with 1 X TBS. Equal volumes of Stable Peroxide Solution and Luminol / Enhancer Solution were mixed, and then diluted 1 : 10 with 1 X TBS. The membrane was incubated in the detection reagent for 5 min and then placed into a clean disposable bag (ROTH). Excess liquid and air bubbles were removed. Detection was performed by exposing the membrane to an X-ray film for an appropriate time. The time of exposure depended on the strength of chemiluminescence, which was influenced by the amount of antigen and bound antibodies. Exposure time was between 10 sec and several minutes. The film was developed in replenisher, washed in water, and fixed in fixer for several minutes. The film was then watered for approx. 15 min and dried.
5.23.6 Testing of Sera
When the properties of different antibodies were to be tested with the same antigen, e.g. with different sera or different dilutions, a preparative gel was used. The gel had only two wells, a small one for the protein size marker and a broad one for the antigen, so the antigen was distributed over the breadth of the gel except for the marker lane. After blotting such a gel, the membrane was cut into small vertical strips, which were incubated in small single dishes with the different antibodies.
These blots were developed using the AP-system. After processing the membrane was reassembled.
5.23.7 Analysis by MALDI-MS
Analysis of the expressed and purified proteins by Matrix Assisted Laser Desorption / Ionization Mass Spectrometry (MALDI-MS) was performed by TOPLAB GmbH. The desired protein band was excised from a Coomassie-stained SDS gel and sent to TOPLAB GmbH. The data were received as an interpreted analysis together with the underlying raw data.
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