I can not find the detection limit for proteins (they say 2ng/ul for dsDNA). Anyone knows? I am introducing changes to my protein that a concentration procedure could alter... and the protein is very diluted, around 2ug/ml. BCA range is between 20-2000 ug/ml, that is why I am exploring the use of take3. Can you suggest me another method for protein quantification at low concentrations? Thank you.
Dear Seidy
I never used the taje3 biotek system but it seems a plate to make biotek readers able to read microvolumes similar to nanodrop.
If you would like to use it for protein quanfication at 280nm you can estimate the detection limit in 2 ways:
1) Experimentally: Preparare a standard solution of bsa eg 10mg/ml. Perform a serial dilution of this sample * eg 5- 2.5- 1.25-0.62-0 31-0.15-0.07 and so on. Read the samples and check which is the minumun concentration tha result in a detectable signal (A>=0.1). Of couse it is not very accurate determination because bsa has a different extinction coefficient respect to your protein but you can have an idea about the magnitude.
2) Theoretically: if you know the lenght of optical path of take3 plates (0.1mm?) And the extinction coefficient of your protein using the lambert beer law you can estrapolate the minimum concentrarion able to result in a detectable assorbance (A>= 0.1)
However my experience with nanodrop suggest that probably you will not able to detect samples with concentration lower that 0.1mg/ml but it is depends also from the ectinction coefficient.
An alternativw simple and very sensitive method for protein quantification using a multiplate reader in fluorescence is nanoorange ( thermo cod .https://www.thermofisher.com/order/catalog/product/N6666)
It is much more sensitive if bca end bradford but to read it you need a fluorescence reader with appropriate filters or a monocromator.
Good luck
Ciao
Manuele
Try Bradfod 1976 mehod which uses Coomassiee Brilliant Blue R250. Commercial kits are available.
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