Lowry Method Anyone?

[discodave]

Hey,

I'm having a little bit of difficulty determining exactly what this assay tells us. At the end of the day, its all about determining protein content, but what does the levels of protein content in our cells tell us.

For example, with cells treated with etoposide, the protein content is supposed to decrease at higher concentrations but what exactly does that mean?

Anyone here doing this sort of thing at university?

 

[.Andy]

I'm having a little bit of difficulty determining exactly what this assay tells us. At the end of the day, its all about determining protein content, but what does the levels of protein content in our cells tell us. For example, with cells treated with etoposide, the protein content is supposed to decrease at higher concentrations but what exactly does that mean?

Hey discodave. The Lowry method is purely a protein quantification method. It really won't tell you anything about your the nature of your cells besides if the protein content has gone up or gone down. Etoposide inhibits topoisomerase 2 (which is involved in separating DNA strands) which in turn inhibits RNA production (as RNA polymerase can't bind and transcribe the genes) and hence will decrease cellular protein production. So in your treated cell population you will have far less protein because topoisomerase isn't doing it's thing which is in turn shutting down the downstream events of transcription and translation. In the bigger picture this means that the cells are in essence being forced to shut down. They can't make protein and hence they can't do their thing, and will eventually die.

A big point to remember is that etoposide will also inhibit DNA replication and hence cell division which is why it is used as a chemotherapeutic. This means that for the purpose of your assay you need to have purified protein from exactly the same number of cells in both the treated and non-treated groups to get a valid comparison. If your cells you are assaying were rapidly replicating, many cells in treatment group will have been killed as they cannot divide which will give you spurious results.

 

[discodave]

.Andy,

Thanks for the information. This helped to clear up some of issues I was having.

David