BS2520 SDS-PAGE Lab Report Introduction This experiment aimed to detect the GLB-1 (B-galactosidase) protein within various adult mouse tissues. The GLB-1 protein is found in mice as an enzyme that catalyses the hydrolysis of ß- galactosides into monosaccharides by breaking the glycosidic bonds (Shayman, 2013). To detect this protein, a variety of techniques were used; firstly, SDS-PAGE. Western Blotting was then used to further separate the proteins. Each protein was then labelled using immunodetection to distinguish the GLB-1 protein from the mixture. The results obtained showed that the molecular weight of the GLB-1 protein had a similar molecular weight in all 5 tissue samples. Methodology Firstly, the protein was prepared for loading. To do this, 20 ul of 2 x loading buffer (65.ffmM Tris-HCI, 2.1% SDS, 26.3% glycerol, 2.5% ß-mercaptoethanol, 0.01% bromophenol blue) was combined with the five tissue samples and were heated to 90°? for 10 minutes. Gel 1 was created by loading 5 ul of the five protein mixtures (100 ul protein was loaded). Gel 2 was created by loading 10 ul of the five protein mixtures (200 ul protein was loaded). One well for each gel contained 4 ul of the marker protein. The electrophoresis was then left to run at 150 V. After 50 minutes, the bromophenol blue dye had run to 1cm from the bottom. Gel 1 was placed in Coomassie stain for one hour. Gel 2 was covered with transfer buffer for 10 minutes. Next the nitrocellulose membrane was prepared. This was done by wetting it in transfer buffer (25mM Tris-HCI, 192mM glycine, 1ff00ml ddH2O, 200ml ethanol). The blotting sandwich was then left in the apparatus for 45 minutes at 100 V. Gel 1 had the stain removed and was covered in destain (20% methanol, 10% acetic acid) and left overnight. Once the blot had finished, the membrane was removed, rinsed with distilled water and left overnight to dry. Gel 2 was placed in Coomassie blue stain and left overnight. Both gels were drained and covered with fresh destain. The membrane was placed in the blocking solution and left on a shaking platform for 30 minutes. The primary antibody solution replaces the blocking solution and was left on the shaking platform for a further 60 minutes to incubate. After, there are 3 x 3 minute washes using TBST buffer (0.02M Tris-HCl, 0.1369M NaCl, 0.1% Tween) which was replaced by the secondary antibody solution; this incubated on the shaking plate for 45 minutes; figure 1 shows a diagram of how the antibodies bind to the GLB-1 protein. The gels were rinsed with water. Next, the wash process with TBST buffer was repeated and the substrate buffer (100mM Tris-HCl, 100mM NaCl, 5mM MgCl2) was made. The membrane was washed
in this buffer twice. It was then left in 10 ml of fresh buffer, combined with ff0 ul of NBT, and left for 20-30 minutes to develop. The development reaction was ceased by washing the membrane in TBST buffer. Emitted chemical signal/ luminescence Secondary antibody (anti- rabbit