Ion Exchange: Chromatograph peak, no protein on SDS PAGE

[DaKdeGO]

Hello everyone,

 

I am curious if you have had this experience before. Currently I am visiting a lab and have been getting really strange results while using their columns for protein purification. The short version is that I've been getting large absorbance peaks (500-2000mAU) on my chromatograph without any protein showing up on my SDS PAGE results. I've had this occur with two different proteins and different different buffers.

 

Any thoughts?

 

Thanks!

[CharonY]

Without looking at those and reference chromatograms (values are too arbitrary to be looked at without reference points) and a look on reproducibility one cannot say too much about it. The peaks could e.g. be contaminations, especially if they show up at the same time. But again, not enough info to troubleshoot.

[BabcockHall]

Are you reading at a single wavelength (280 nm), or are you taking spectra?

[melD09]

Your protein may be too dilute to be detected on the gel, the collected fraction should be concentrated before loading on the gel if possible. You don't specify the column type or buffer system, but some buffer components absorb in the UV, for example imidazole.