Trouble shoot with radioactive Enzyme activity assay - (Jan/18/2013 )

I am having problems with my assays, my blanks, which only contains water instead of the enzyme i am using, reads often in thousands of counts( in CPM), even when i leave them overnight to avoid chemo-luminescence. I am not sure where i went wrong in the process because this is not consistent, sometimes i get around hundreds and sometimes below 100, which is the desired value.

What really troubles me is that i often get my activities around thousands as well, usually in between 5000 to 15000 depending on the concentration and the amount of the enzyme i add. So if my blanks are at the value of thousands, say 2000, from my last assay. I am not even sure if i should trust my readings as real enzymatic activities.

I use H3_glutamate

please give me some insights and suggestions.

Cheers

A bit more on your protocol would be helpful so that we can see if there is something wrong in the way you are doing things.

Could it be that you are contaminating the scintillation counter with something on your gloves?

Sometimes your radioactive substrate spontaneously forms the product, this is what happened in my case. But the conversion of substrate is more when you add enzyme. Therefore to avoid this problem the radioactive counts that we got from spontaneous reaction were decreased from the enzymatic reaction counts. That way you relate your enzymatic reaction to the blank and you get accurate results.

how do you separate the product from the substrate?

My radioactive assays were based on TLC so there you can see the conversion of substrate into product as both run on different Rf.

letting the vials sit overnight to reduce or eliminate chemiluminescence is good but sometimes it's static on the vial that causes extra counts. we wipe our vials with a wet kimwipe to eliminate static (some scintillation counters have static eliminators but they don't always work perfectly). the static can be caused by handling the vials with latex gloves.

neuron on Wed Jan 23 03:58:49 2013 said:

I don't know if its the same. But detection method is different. mimiw is taking counts of radioactivity by scintillation counter which contains both substrate and product. Whereas in my case, we are separating product and substrate on TLC and then taking counts. Since we take radioactive substrate, in blank there should not be any product. But in blank also we see some product.