It has been a no good awful very bad day in your lab, and you making all sorts o

Question

It has been a no good awful very bad day in your lab, and you making all sorts of mistakeswith this Western Blot you are trying to do.

a. You realize that your primary antibody was generated in mice, but the only secondaryantibody you have is targeted against goat antibodies. Will your Western blot work if

you use this secondary antibody? Why or why not?

b. Rather than risk it, you borrow an antimouse secondary antibody from the lab nextdoor. Upon seeing the results, you find that, instead of a nice sharp band or two, thewhole blot has “lit up.” What did you do wrong to cause the whole blot to light up?

c. You go back and rerun the Western blot, being sure not to make the same mistake.Now instead of having the whole blot light up, you see a set of clear bands on the gel,but rather than being one band per lane at a given size, you see two bands (about 10kDa different in size) in each lane. There are several possible explanations for thisphenomenon, some trivial, and some interesting. Give at least two possible reasons forthis pattern of bands.

Explanation / Answer

Answer:

a. Secondary antibodies are usually well developed and show strong binding to the intended primary antibody with minimal background. A secondary anti-goat should not bind to mouse antibodies. Of course this does not eliminate the risk of cross-reactivity completely, but the chance that the secondary antibody cross-reacts with the protein of interest is not any higher than the chance that it interacts with any other protein present in the extract.

b. There may be several factors that resulted in the whole western blot to lit up:

i. Problem with the blocking step or if the blocking step is skipped

ii. Use of low fluorescence PVDF membrane for the LICOR system of developing western blots.

iii. Excess Secondary antibodies may lead to a background signal.

iv. Not enough washing in between incubation steps

v. If SDS is not added to the secondary antibody solution (0.01 – 0.02%).

c. The formation of this type of band patterns in the western blot can be due to the following reasons:

i. Higher molecular weight bands are often seen when the target protein is postranslationally modified. Acetylation, methylation, myristoylation, phosphorylation, glycosylation and ubiquitination are all modifications that increase the molecular weight of a protein.

ii. If too much lysate is loaded onto a gel, antibodies can bind non-specifically to proteins of excessive abundance, resulting in more than one band.

iii. Degradation of the protein due to specific cleavage by a protease.

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