Question

You are performing a mobility shift assay with a protein complex that is composed of proteins A, B, C, & D, each of which can bind to the DNA of interest. You run the gel with the lanes as follows:

Lane 1: Protein A + DNA
Lane 2: Proteins A + B + DNA
Lane 3: Proteins A + B + C + DNA
Lane 4: Proteins A + B + C + D + DNA
In which lane will the DNA run closest to the top of the gel?

Answer 1

In a mobility shift assay, DNA will run closest to the top of the gel in the lane where it is complexed with the most proteins. In this case, Lane 4, with DNA bound to Proteins A, B, C, and D, will display the DNA closest to the top due to the increased size and reduced mobility of the complex.

Step-by-step explanation:

In a mobility shift assay, also known as an electrophoretic mobility shift assay (EMSA), the migration of DNA through a gel is affected by its interaction with proteins. Typically, the more proteins that are bound to the DNA, the slower the DNA will migrate through the gel because the resulting DNA-protein complex will be larger and more complex, causing it to move more slowly compared to smaller DNA-protein complexes or free DNA.

Therefore, in the given experiment:

• Lane 1 contains DNA bound to Protein A only.
• Lane 2 contains DNA bound to Proteins A and B.
• Lane 3 contains DNA bound to Proteins A, B, and C.
• Lane 4 contains DNA bound to Proteins A, B, C, and D.

As we add more proteins, the size and complexity of the complexes increase, making them migrate more slowly during gel electrophoresis. Thus, the DNA in Lane 4, which is complexed with the largest number of proteins (A, B, C, and D), will run closest to the top of the gel due to its larger size and reduced mobility relative to the other lanes.