hi,
I am purifying the FHF2 protein by, Ni-NTA column chromatography followed by heparin column chromatography. After purification I have loaded the purified sample in SDS page gel, there is a single band of 27kda. When I measure the absorbance of purified protein by UV spectroscopy, I am getting two max absorbance peaks one at 230 nm and the other at 277nm. what would be the impurity at 230 nm? please help me out.
The absorbance from 230 nm can be from the peptide bonds.
It is a quite standard feature. If the protein contains aromatic residue then an additional peak around 270 is observed. As mentioned by Marius 230 nm peak is of peptide bonds.
Article UV–Vis spectroscopy of tyrosine side-groups in studies of pr...
http://omlc.org/spectra/PhotochemCAD/html/091.html
http://what-when-how.com/molecular-biology/absorption-spectroscopy-molecular-biology/
You are not observing impurities, you are looking at a single spectral signal which is normal. The absorbance is due to the types of molecule arrangements and bonding. Please read up on some basic chemistry and spectrophotometer use to measure peptides and proteins (Tuhin above has some examples for you).
As you appear to be a student unfamiliar with chemistry, please ask your course instructors or teachers for reference sources or material (books) to help you.
I was surprised because the absorbance maximum at 280 is 0.4, but for 230 it is 2
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