Imidazole contribute to 280nm reading.
Your case probably is because the amount of imidazole in your blank is higher than the amount of imidazole in your eluted protein sample. For an accurate reading you should make sure that the amount of imidazole in the blank is equal to the amount of imidazole in your eluted protein sample.
Hi
you have measured the absorbance at 280nm which is sensible for Proteins containing aromatic amino acids. it's clear that your
protein didn't contains many aromatic acids (trep or phenylalaline). that's why it doesn't absorb at 280nm.
Removed imidazole as well but still no peaks at 280nm. I am running another gel to see whether I can see the bands again.
Hi Muhammad,
I would recommend that your dialyse your sample to remove Imidazole before measuring the concentration on the nanodrop.
in addition to that, carrefully check the aromatic amino acid contain of your protein and make sure that your enter the right molar extinction coefficient.
something you can try also is a BCA assay whih is based on the absorption at 562nm of a product resulting of a reaction between your protein and other reagents. ( in the case your aprotein absorption at 280nm is too low)
Good luck.
As already discussed A280 depends entirely on the presence of aromatic a.a.s. If you have the full sequence of your protein try putting it into Protein Calculator to get a theoretical extinction co-efficient:
http://protcalc.sourceforge.net/
it may not be accurate for a fully folded protein but it may give you some idea of what to expect.
(1) Check your amino acid content for side-chains which absorb at that wavelength: tryptophan (W), tyrosine (Y) and cysteine (C) (or use something like http://web.expasy.org/protparam/ which will give you a predicted extinction coefficient)
(2) Make sure to remove the imidazole from your sample either via a buffer exchange column or dialysis. High concentrations of imidazole will skew any data as the overall absorbance will be too high and out of the range of detection.
(3) Test your nano-drop with a different protein, nano-drops can break down or might need a good clean as the sensor area is very small.
(4) If you have access to a spectrophotometer, try using it to detect your protein instead, as these instruments tend to be much more sensitive.
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