Western blot experiments

Fig. 2. Illustration of the setup of the sandwich cassette in the transfer tank observed from the top for the western blotting experiment.

B 10. In your biochemistry research project, you have isolated a new protein from spinach leaves. You wish to do a Western blot experiment with the protein to help determine its chemical structure and/or biological function. An amino acid analysis of the protein showed a great abundance of phenylalanine, leucine, and valine. What would be your choice of membrane for the blotting experiments ... 

S. Farrell and L. Farrell,/. Cbem. Educ. 73, 740-742 (1995). A Fast and Inexpensive Western Blot Experiment for the Undergraduate Laboratory. ... 

MCAo. Western blotting experiments demonstrated that these early effects were associated, 24 h after permanent MCAo, to a significant increase in the phosphorylation and activity of the prosurvival kinase, Akt. Indeed, BEO significandy enhanced the phosphorylation of the deleterious downstream kinase, GSK-3/3, whose activity is negatively regulated via phosphorylation by Akt. 

At this point, the anti-/3-galactosidase antibody (primary antibody) diluted in blocking buffer is added to the well. During the incubation, it will bind to the /3-galactosidase adsorbed to the bottom of the well. Several washes are then performed to remove primary antibody that may have bound nonspecifically to proteins on the bottom of the well other than /3-galactosidase. As in the Western blot experiment, the wash solution contains a small... 

Antibodies can be used for a variety of applications in the molecular characterization of receptors and receptor-hgand interactions. Antibodies can be used for the detection of receptors in tissue shces. Western blot experiments [48], or ELISAs (enzyme-linked immunosorbent assays) [49]. They can also be used in competition experiments to map the binding epitope of a hgand [50]. Even though the use of antibodies is routine, fhere is no general protocol for fheir generation. 

Lox-1 and Iox-2 were separated after hydroxyapatite chromatography. Two activity peaks were detected, the first eluting at 79 mM potassium phosphate, the second after 102 mM. Product analysis showed that the first activity peak almost exclusively contained lox-1, as it mainly formed 9-HPOD after incubation with linoleic acid. The second peak mainly contained lox-2. Western blotting experiments confinned the separation of both isoenzymes by this single column step (data not shown). 

---