protein concentration measurement - (Nov/27/2008 )

Dear all
I have a protein without any tryptophan, tyrosin or cystein.
Whats the best method to measure protein concentration ?
My protein has strong absorption at 214nm, my photometer has an option to measure at 205nm, I am afraid that is not to accurate enough.
I was told BCA is also not reliable in this case.
Does anybody have another idea ?
pET

we normally do Bradford assay on 96-well plates. it's so easy, but I'm not sure it can be used in your case.

you can try the lowry or microbiuret protein assay (both detect peptide bonds, destructively) or the bradford (dye binding). make sure you have an appropriate standard and proper blanking.

214 or 205nm can be used to read peptide bonds but are also interfered with by buffer components. you will need to use a buffer that exhibits minimal absorbance at the wavelength you choose. also, choose an appropriate standard and blank properly.

[quote name='mdfenko' date='Dec 1 2008, 06:26 PM' post='159075']
you can try the lowry or microbiuret protein assay (both detect peptide bonds, destructively) or the bradford (dye binding). make sure you have an appropriate standard and proper blanking.

214 or 205nm can be used to read peptide bonds but are also interfered with by buffer components. you will need to use a buffer that exhibits minimal absorbance at the wavelength you choose. also, choose an appropriate standard and blank properly.

Thank you mdfenko for your reply.
If I measure the protein with 214nm on my photometer do you know the factor to calculate the protein concentration [conc = F x absorbtion] ?
pET

the response depends on the extinction coefficient of the specific protein.

but,

if you have a suitable standard curve then you can determine the change in absorbance per microgram protein and apply that to determine your protein concentration.