I was working with GE superdex 200 column on AKTA prime plus system. I cleaned my column with NaOH, water and then I equibrilated it with buffer. During purification I saw no change in UV, it was running straight at zero. And I collected purified protein of my interest in fractions. When I ended my program, I did autoscaling for UV. Chromatogram that I saved on system after purification looks totally different than what I was observing during purification. This might be something very silly but I'm really not an expert for AKTA at least for now. So it would be a great help if you could please help me in understanding and resolving this problem.
My saved chromatogram after purification looks scary. I have attached a picture for your reference with both chromatogram.
Thank you.
I could not see the scale of the vertical axis from your saved chromatogram. Did you set your UV at 280 nm during purification and when you save the chromatogram? Did you run your protein sample on SDS-PAGE prior to purification? Were you able to detect your target proteins on the gel? What was your protein sample prior to purification? Is it from cell lysate/tissue homogenates? Is it a partially purified protein by other techniques? Is your sample prepared freshly? Stored at 4, -20 or -80 degree for sometime prior to purification? Did you centrifuge freeze-thawed solution prior to purification? Did you observe insoluble aggregates after centrifugation?
I have speculated that either you perform purification and saved the chromatogram with the incorrect wave length or the concentration of your protein was very low in the solution. You need to consider dilution of your samples during purification by the running buffer.
Hi Alemu,
Thank you for adding your answer. My loading sample was taken after NiNTA affinity purification . And yes I checked my protein prior to (concentrated up-to 4mg/ml) and after size exclusion purification on SDS PAGE gel. I have my protein where it is supposed to elute on Superdex 200. I had no aggregates and it's freshly taken with no freeze thawing etc. Possibly it was run on incorrect wavelength. While saving and autoscaling UV, I got no option about changing wavelength range. And I'm not sure how can I work on unicorn to look for such data. Or is it something to be checked on instrumentation side?
I know a wavelength range thing is there on the side of instrument. But I never touched it. I definitely need to check there.
You can adjust it using the Unicorn 7 control software for AKTA protein purification systems. You can check the manual how to use the software. I think it is possible to get some tutorial videos for that. You can ask experts from GE.
I hope you will fix your problem. Good luck!
The UV absorption is very low and only after zooming in you are getting the peak. Either the system is very old and lamp life is at the end (can be checked on the system itself) or the UV filter is dirty, which can be changed to other wavelengths and an increase in UV response will be observed. Usually there are more than one filter like an additional 254 nm filter besides 280 nm.
Hi there,
As mentioned above by Mangal, there might be an issue with your detector. To get a clearer opinion you should analyze the peak elution fractions by SDS PAGE (and maybe just UV measurement would tell you) : if there are bands, there is definitely an issue with the detection ; if no clear bands, your sample might have precipitated somewhere between NiNTA and the AKTA detector.
The difference is obviously in y-axis scale. While during purification it was set to 3.0, which is way too high, after saving it auto-scaled. I think you can set up for it to auto-scale during purification (or at least set the maximum to something like 0.5 or 0.1).
Anyway, you need to determine the amount of recovered protein to determine, whether it's problem with the system or with your sample.
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