Isopropanol

SDS-Polyacrylamide gels were prepared and the glass plates were washed with 70% ethanol and water. After drying the plates, water was used for test leakages. Two SDS-Polyacrylamide gels were prepared according to the following recipe.

These all above components of the running gel were added in a 50 ml tube and solutions were mixed and pipetted into the prepared gel chambers. Glass plates were filled ¾ and the gel was covered with 100-500 µl Isopropanol in order to achieve an even surface. When the gel was polymerized, isopropanol was removed on the top of the running gel with water and thus, water was also removed as well. Then, stacking gel was prepared and its polymerization reaction was started with TEMED, and its solution was quickly poured on the top of the running gel. Therefore, a comb was inserted into the stacking gel before its polymerization, to form the loading wells. When the gel was polymerized, the inner chamber was filled with electrophoresis running buffer and the outer chamber was filled half.
Chemical modification of ERAB (Wild Type and Mutant) Protein with Fluorescein Maleimide
Wild type and mutant ERAB

The gel piece with the lanes 7-10 was put in a 10 ml blotting buffer until western blotting. The gel piece containing the lanes 1-6 was then placed under UV-light or onto a transilluminator for the visualization of the fluorescent modification. Then the visualized gel was documented by taking a picture in Gene map program. Afterwards, the visualized gel was stained with Coomassie Brilliant Blue protein stain. Staining was done for 30 to 60 mins, within this time the staining solution was recycled after 15 mins. Then destaining was done in a destaining solution and the solution was recycled several times until the background staining disappears and the used solution was collected in a bottle. Then its picture was