Protein concentration assays

Go to the profile of nekameneka
Jan 27, 2017
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Hi everyone,

I have question about interference during protein concentration assays. I have my protein purified but my sample also contains 5 mM ATP during the experiment. I am quiet sure that ATP interferes with Nanodrop when I want to check concentration of my protein in the end. I expect low concentration and I get extremely high values (even if I blank the Nanodrop with only buffer containing ATP). My question is how can I find out concentration of my protein? Does ATP interferes with Bradford? I searched and I found mixed information.
Thank you

5 Comments

Go to the profile of mdfenko
mdfenko 7 months ago

according to bio-rad, their "bio-rad protein assay" used for the standard assay is compatible with up to 1 mM atp. thermopierce does not state any compatibility with atp with their bradford protein assays.

Go to the profile of nickk2017
nickk2017 7 months ago

hi
There is no single protein assay method that yields absolutely accurate results. Each method has different advantages and limitations.
The Pierce 660nm assay is the best. Just add protein to the reagent and measure 660nM. I used in in purification of membrane proteins solubilized with different detergents. See the Pierce catalogue and read about it. It is also,compatible with reducing agents. I have used it with DTT, TCEP, and betamercaptoethanol.
It is cheap too.


Thanks

Go to the profile of nekameneka
nekameneka 7 months ago

Thank you for your suggestion.
I also have another question. I am trying to calculate my protein in molar concentrations instead of ug/ul, there are so many different explanations. Can someone give me a formula and explain how to calculate for example how much ug/ul is 40uM???
Thanks a lot!

Go to the profile of mdfenko
mdfenko 7 months ago

the molar concentration depends on the molecular weight (mw or fw) of the protein. you can't just say that xug/ul=40uM.

an example using bsa:

mw of bsa is ~67kDa (67,000 grams/mole). 1M=67,000g/liter, 1mM=67g/liter, 1uM=67mg/liter=67ug/ml=67ng/ul---40uM=2680ng/ul=2.68ug/ul

Go to the profile of nekameneka
nekameneka 7 months ago

I know I need MW of the protein but there were so many explanations, including online conversions, who also asked for the volume of the sample, didn't quiet understand why they need volume. Anyway, thank you for a simple explanation, I understand