Unfortunately 220 nm is a difficult wavelength to measure protein
concentration at.
The thing you must note before assaying proteins is that every (at
least every one I am familiar with) assay is prone to a large error
margin because they are dependant on amino acid makeup of the protein.
At 280 nm you can assay for tryptophan (be sure to use a quartz
cuvette). The Beer-Lambert Law states:
log (absorbance) = (molar extinction coefficient in liters per
mole-centimeter) * (concentration of absorbing species) * (path length
in cm)
from this, you can either look up the exctinction coefficient for
tryptophan in the CRC handbook or some other source, or use a sample
of known tryptophan concentration to calculate it.
There are a number of other assays, but what it comes down to is that
they will be incredibly innacurate so far as finding a molar
concentration because proteins vary in size dramatically. In order to
get an accurate concentration you are going to have to first purify
the protein you are assaying for because proteins vary dramatically in
size. This UV assay is definitely the worst assay available, so if
you have the capability to do these other assays they will give you
far better information.
These assays include:
The Lowry Assay
There are many different ways of making the Lowry assay reagents, but
once you have them the assay is:
Take 800 microliters of sample
Add 800 microliters of Lowry concentrate, mix, and incubate at room
temp ten mins
Add 400 microliters 0.2N Folin reagent, and vortex
Incubate at room temp 30 mins
Read absorbance at 750 nm
Bradford Assay:
mix 0.04 ml sample with 2 ml assay reagent
Read absorbance at 595 nm
Enjoy. |
