It might be imidazole in case you use it to elute the protein from
Ni-sepharose. Thus, either use the buffer with imidazole for reference, or,
more correctly and accurately, desalt you protein by dialysis against the
sample buffer or by ion-exchange chromatography.
"B.H.Kang" <heony at postech.ac.kr> wrote in message
news:92u66t$a4n$1 at news.postech.ac.kr...
> I prepared the protein with histidine tag in bacterial cell and purified
> with Ni resin.
> For the quantitation of my protein, I check the UV absorption.
> Surprisingly, the maximun absorbance was at 260 nm instead of 280 nm
> and the absorbance was slightly increased comparing the real concentration
> of the protein.
> Is this usual case?
> I think that my protein has no significant nucleic acid contamination.
> And the buffer contains 1 mM DTT,
> The sample buffer was used for baseline of UV absorbance.
>> I didn't know the reason...
>> Thanks and happy new year...!
>>
