How I could determine the extinction coefficient (Molar absorptivity) of my protein experimentaly?
Is it okey if I calculate the slop of Absorption vs Concentration? for this I must prepare some standard dilutions of my protein. is it need to determined each dilutions concentration with bradford, too?
Indeed the slope of your absorption spectrum would be your extinction coefficient as long as your pathlength is fixed (according to Beer-Lambert law) and you can accurately determine the concentration of each sample. Bradford can be problematic as it gives different results for different proteins and hence its worth checking with other kits too. You can also determine the concentration of your protein using refractometry.
Additionally, there is another strategy: You can use ProtParam (http://web.expasy.org/protparam/) to calculate the extinction coefficient of your protein based on the primary sequence. This extinction coefficient is valid for your protein in a denatured state as this does not take into account any structural information, but merely the amino acid sequence. If you measure the absorbance of two samples (one in native and one in denaturing conditions, like 6 M guanidine hydrochloride) with exactly the same protein amount, you can calculate the extinction coefficient of your native protein using the different absorbance values of the two samples and the extinction coefficient of the denatured protein (from ProtParam) as such:
( Anative / epsilonnative ) = ( Adenatured / epsilondenatured )
This is based on the Beer-Lambert law ( A = epsilon * concentration * pathlength) where the concentration and measurement pathlength of the two samples are the same.
Hope this helps,
Arne
Indeed the slope of your absorption spectrum would be your extinction coefficient as long as your pathlength is fixed (according to Beer-Lambert law) and you can accurately determine the concentration of each sample. Bradford can be problematic as it gives different results for different proteins and hence its worth checking with other kits too. You can also determine the concentration of your protein using refractometry.
Additionally, there is another strategy: You can use ProtParam (http://web.expasy.org/protparam/) to calculate the extinction coefficient of your protein based on the primary sequence. This extinction coefficient is valid for your protein in a denatured state as this does not take into account any structural information, but merely the amino acid sequence. If you measure the absorbance of two samples (one in native and one in denaturing conditions, like 6 M guanidine hydrochloride) with exactly the same protein amount, you can calculate the extinction coefficient of your native protein using the different absorbance values of the two samples and the extinction coefficient of the denatured protein (from ProtParam) as such:
( Anative / epsilonnative ) = ( Adenatured / epsilondenatured )
This is based on the Beer-Lambert law ( A = epsilon * concentration * pathlength) where the concentration and measurement pathlength of the two samples are the same.
Hope this helps,
Arne
I would add to Arne Raasakka's excellent answer a warning about measuring the UV absorbance of the native protein. If the sample contains any aggregated material, it will elevate the UV absorbance due to light scattering, giving a false high value. You can check for aggregation by dynamic light scattering.
If the protein contains any tightly or covalently bound ligands or prosthetic groups, they will add to the absorbance spectrum. In that case, it is essential that their stoichiometries be known and reproducible from prep to prep.
Finally, it is essential that the protein be of high purity and free of other absorbing substances in order to get an accurate and reproducible result for the extinction coefficient.
for Nacl is it 0.69 the value validated for path lenght of 1mm film electrolytic.
the regression equation can then be more valuable for other concentration in the same way. Can everybody help me for this because in the litterature there are another concept (proteines ...).
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