Question

During the last week of the lab (week 11), you "performed" a Western blot comparing the levels of myosin and tubulin between C2C12 myoblast (Mb) and myotube (Mt) extracts. The SDS-PAGE was loaded with two sets of Mb and Mt lanes and transferred onto a PVDF membrane. The membrane was then cut out into two pieces, with each piece having a pair of Mt/Mb lanes, allowing each piece to be incubated with a different primary antibody. A rabbit IgG antibody was used with one piece of the blot to detect myosin and a mouse IgG antibody was used on the second piece of the to detect tubulin. Each piece of the blot was then incubated with an HRP-tagged secondary antibodies specific to each primary antibody. Select the blot pair that would best represent the expected results of your WB experiment.

Answer 1

Myoblast (Mb)

Myotube (Mt)

Western Blot # 1

Myosin

Primary antibody: Rabbit IgG Anti-myosin

Secondary antibody: Anti-Rabbit myosin HRP conjugate

Western Blot # 2

Primary antibody à anti-tubulin Mouse IgG Tubulin

Secondary antibody: Anti-mouse tubulin HRP conjugate

Since, myosin is not that much expressive in myoblast and myotube as compared to tubulin. Hence, best result can be seen in western blot #2.

Primary anti-body Mouse anti-tub IgG, anti mouse tub (HRP conjugate) and band can be visualized through gel documentation system.