HPLC peaks with my buffer alone..

[Genepro]

Hello all,

I am trying to purify and concentrate my Histidine tagged protein that was eluted off of Ni-NTA column. The eluted protein is present in the elution buffer with the following composition

20mM Tris-HCl
0.5M Nacl
0.5M Imidazole, pH 8.0

When I analyzed a few microliters of my protein (in elution buffer) on an SDS-PAGE, it gave me a band at the right size.

Here comes my HPLC related questions.

1) I used 1ml of my elution buffer alone (without any protein in it) on a C-18 column attached to HPLC. I used 100% water to elute the buffer out (A-water and B-Acetonitrile). When I looked at my chromatogram, I found 2 peaks in both 214nm and 280nm plots. This is usually observed when a protein or a peptide elutes. But how did it so for a buffer alone?

2) When I used 1ml of my eluted protein (in elution buffer) under the same run conditions, it gave me the exactly the same kind of peaks as for the buffer. After my wash with 100% water, I tried running 0-100% acetonitrile gradient to see whether my protein that was stuck to the column might elute. But I couldn't find any.

Can anybody help me on this kind of behavoir? Do I have to dialyze my protein and then do a HPLC??

Waiting for your advice...Thanks in advance

Genepro

Any suggestions or advice is welcome.

[HW Mueller]

I can only give some general advice:
Your buffers absorb strongly at 214 nm, TRIS absorbs fairly at 280 nm, I wouldn´t be surprised if imidazole also did. From the examples which I have seen one needs more than H2O and ACN to get proteins off a C-18 column. One usually needs pH and ion strength control, chaotropes, or detergents besides organics. Usually column manufacturers have some examples, or books on protein handling. "Proteomics" is a good key word in general searches.