[Messi]

Hi,

I am looking at results of a TRAP assay (Telomerase inhibition) and have a tough time understanding the gel electrophoresis being shown. I was wondering if someone could enlighten me on the results please?

I have shown the gel below, with each lane being labelled as different concentrations of the ligand + telomere.

I know what the IC₅₀ values were determined to be based on the electrophoresis data, but I was unsure how it was determined.

I hope someone can enlighten me!

[Yggdrasil]

Here's a paper describing the TRAP assay: https://www.sciencedirect.com/science/article/pii/S0009898106001549

[Messi]

Here's a paper describing the TRAP assay: https://www.sciencedirect.com/science/article/pii/S0009898106001549

Thanks Yggdrasil. I just had a hard time understanding the gel. Do you think you could explain to me the gel results in two or three lines if you don't mind?

[Yggdrasil]

Basically, telomerase adds 6 nucleotides at a time to the end of a DNA primer.  After it adds 6 nucleotides, it can either fall off or repeat its enzymatic cycle and add 6 more nucleotides.

Thus, when you look at the products of a telomerase reaction, you will see the unreacted primer at the very bottom of the gel, and above that band you will see a series of bands corresponding to primers that have been extended by the enzyme.

Although it's hard to tell how the authors measured the activity of the enzyme without looking at the paper, my guess is that they measured enzyme activity in each condition simply by quantifying the fraction of the DNA that got extended (i.e. the fraction of signal in all the bands except for the bottom band).

[Messi]

Basically, telomerase adds 6 nucleotides at a time to the end of a DNA primer.  After it adds 6 nucleotides, it can either fall off or repeat its enzymatic cycle and add 6 more nucleotides.

Thus, when you look at the products of a telomerase reaction, you will see the unreacted primer at the very bottom of the gel, and above that band you will see a series of bands corresponding to primers that have been extended by the enzyme.

Although it's hard to tell how the authors measured the activity of the enzyme without looking at the paper, my guess is that they measured enzyme activity in each condition simply by quantifying the fraction of the DNA that got extended (i.e. the fraction of signal in all the bands except for the bottom band).

Beautifully explained! Thanks a ton!